Methods of assessing and treating cancer in subjects having dysregulated lymphatic systems

ABSTRACT

Provided herein is a method for determining cancer treatment using an immune-modulating therapy in a subject in need thereof. The method comprises assessing whether a lymphatic system in a subject is dysregulated. When the lymphatic system is dysregulated, a treatment for the lymphatic system is determined before a therapeutic amount of an immune-modulating therapy is administered to treat cancer in the subject. Alternatively, when the lymphatic system is dysregulated, an immune-modulating therapy is selected to treat cancer in the subject. The immune-modulating therapy is independent of immune-cell priming, antigen trafficking, antigen presentation, and any combination thereof. The subject may also be treated for cancer accordingly.

This application is a continuation application of U.S. Ser. No.16/155,726, filed Oct. 9, 2018, now allowed, which is a continuationapplication of PCT/US2017/053504, filed Sep. 26, 2017, published as WO2018/058125 on Mar. 29, 2018, which claims the benefit of priority ofU.S. Provisional Application No. 62/412,488 filed Oct. 25, 2016, andU.S. Provisional Application No. 62/399,766 filed Sep. 26, 2016, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

This disclosure relates to the field of diagnosing and treating cancer,particularly cancer in subjects having dysregulated lymphatic systems towhom an immune-modulating therapy is applied.

Patients who suffer from a dysregulated lymphatic system do not respondto immune-modulating therapies, which depend on immune cell priming,antigen presentation, or antigen trafficking. Patients with tumorstreated with cancer immune-modulating therapy do not respond. By notresponding, patients specifically rapidly progress from their tumor withminimal to no response period. Similarly, such patients have extremelypoor overall survival compared to their counterparts who do not havedysregulation or dysfunction of their lymphatic system. Also, patientswith lymphangitic carcinomatosis or lymphatic invasion do not respond tosuch cancer immune-modulating therapies, for lack of immune cellactivation and immune cell priming. A mouse melanoma model has shownthat transgenic animals born without lymphatics do not have local tumorimmune infiltrates, specific CD8 T cells, and antigen-presenting anddendritic cells in the tumor sites.

Taken together, prior studies examining the effects of blocking VEGF-Cand its receptors on tumor metastasis have examined the effects ofdifferent antagonists on preventing metastatic spread of the primarytumor. But these prior studies have not discussed the effects of suchantagonists on the progression of established distant metastases afterremoval of the primary tumor. What is critically needed in the art arecompositions and methods for achieving the treatment of establishedmetastatic disease in cases when primary tumors have been removed or arenon-resectable.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification and a study of the drawings.

The following embodiments and aspects thereof are described andillustrated in conjunction with compositions and methods, which aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

Provided herein is a method for treating cancer in a subject in needthereof. The method comprises, when a lymphatic system in a subject isdysregulated, administering to the subject a therapeutic amount of adrug to regulate the dysregulated lymphatic system, and administering tothe subject a therapeutic amount of an immune modifying therapy.Alternatively, when the lymphatic system is dysregulated, a therapeuticamount of an immune-modulating therapy is administered to the subject,which immune-modulating therapy operates independently of immune-cellpriming, antigen trafficking, antigen presentation, and any combinationthereof.

Also provided herein is a method for determining cancer treatment usingan immune-modulating therapy in a subject in need thereof. The methodcomprises assessing whether a lymphatic system in a subject isdysregulated. When the lymphatic system is dysregulated, a treatment forthe lymphatic system is determined before a therapeutic amount of animmune-modulating therapy is administered to treat cancer in thesubject. Alternatively, when the lymphatic system is dysregulated, animmune-modulating therapy is selected to treat cancer in the subject,which immune-modulating therapy is independent of immune-cell priming,antigen trafficking, antigen presentation, and any combination thereof.

Additional embodiments and features are in part in the description thatfollows, and in part will become apparent to those skilled in the artupon examination of the specification or may be learned by the practiceof the embodiments discussed herein. A further understanding of thenature and advantages of certain embodiments may be realized byreference to the remaining portions of the specification and thedrawings, which form a part of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be readily understood by the following detaileddescription in conjunction with the accompanying drawings, wherein likereference numerals designate like structural elements. The drawingsprovide exemplary embodiments or aspects of the disclosure and do notlimit the scope of the disclosure.

FIG. 1 is a schematic of general lymphatic biology showing moleculesthat modulate tumor lymphangiogenesis. See Stacker et al.,“Lymphangiogenesis and lymphatic vessel remodeling in cancer,” NatureReviews Cancer, 14:159-172 (2014), incorporated herein by reference.

FIG. 2 is a schematic showing The VEGF family of ligands and theirrespective binding patterns to the VEGFR. See Karkkainen et al.,“Lymphatic endothelium: a new frontier of metastasis research,” NatureCell Biology, 4:E2-E5 (2002).

FIG. 3 shows progression-free survival (%) as a function of time in daysfor cancer treatment using immune modifying therapy. Biomarker positivepatients had a median survival of 50 days. Biomarker negative patientshad a median survival of 151 days. HR for progression or death was 0.48(95% CI, 025.-0.91), at P<0.025.

FIG. 4 shows the objective response of partial responses or completeresponses (PR/CR). The first partial responses were denoted by ellipses.The solid triangles show ongoing responses. Biomarker negative patientshad an N=18/95 (19%). Biomarker Positive patients had an N=0/95 (0%).

FIG. 5 shows the durable clinical response for the stable disease (SD)or partial response (PR) lasting at least 180 days. Lines 1-26 werebiomarker negative patients. Line 27 was a biomarker positive patient.

FIG. 6 shows overall survival (% alive) as a function of time in days.Biomarker positive patients survived a median of 47 to 67 days.Biomarker negative patients survived a median of 445 days.

FIG. 7 shows progression-free survival (%) as a function of time indays. Biomarker positive patients had a median survival of 47 to 68days. Biomarker negative patients had a median survival of 189 days.

DETAILED DESCRIPTION I. Methods for Determining Cancer Treatment

Provided herein is a method for determining cancer treatment using animmune-modulating therapy in a subject in need thereof. The methodcomprises assessing whether a lymphatic system in a subject isdysregulated. When the lymphatic system is dysregulated, a treatment forthe lymphatic system is determined before a therapeutic amount of animmune-modulating therapy is administered to treat cancer in thesubject. Alternatively, when the lymphatic system is dysregulated, animmune-modulating therapy is selected to treat cancer in the subject,which immune-modulating therapy is independent of immune-cell priming,antigen trafficking, antigen presentation, and any combination thereof.

Molecules that modulate tumor lymphangiogenesis are shown in FIG. 1,with soluble ligands presented outside the cell, cognate receptors atthe cell surface, and transcription factors in the nucleus. Vascularendothelial growth factor C (VEGFC) and VEGFD refer to theproteolytically processed, biologically active forms of these proteins.Most ligands shown promote lymphangiogenesis, while transforming growthfactor-β (TGFβ) inhibits lymphangiogenesis. Other molecules are known toparticipate in lymphatic development in the embryo, such as collagen andcalcium-binding EGF domain-containing protein 1 (CCBE1; not shown), forwhich a role in tumor lymphangiogenesis has not been shown. Theinteraction of tumor cells with lymphatic vessels can be promoted byinterstitial fluid flow (which partly results from lymphatic drainage)via autologous chemotaxis involving chemokines, such as CC-chemokineligand 21 (CCL21), and their receptors (CCR7 in the case of CCL21),expressed by tumor cells. Expression of CCL21 on lymphatic endothelialcells (LECs) can promote the entry of tumor cells into lymphatics via aCCR7-dependent mechanism. Producing lymphangiogenic growth factors, suchas VEGFC and VEGFD, can drive the formation of new lymphatics andlymphatic enlargement near a tumor, which increases the surface area forthe interaction of tumor cells with lymphatics. VEGFC can also promotetumor cell invasiveness in an autocrine manner, and it can upregulatethe production of CCL21 on lymphatic vessels. The other abbreviationslisted are 15-PGDH, 15-hydroxyprostaglandin dehydrogenase; ANGPT2,angiopoietin 2; COUPTF2, COUP transcription factor 2; COX2,cyclooxygenase 2; CSF1, colony-stimulating factor 1; EGF, epidermalgrowth factor; EGFR, EGF receptor; EPO, erythropoietin; EPOR, EPOreceptor; FGF, fibroblast growth factor; FGFR, FGF receptor; FOXC2,forkhead box protein C2; PDGF-BB, platelet-derived growth factor BB;PDGFR, PDGF receptor; PROX1, prospero homeobox protein 1; RAMP2,receptor activity-modifying protein 2; SP, sphingosine-1-phosphate;TGFβR, TGFβ receptor; VEGFR, VEGF receptor.

The VEGF family of ligands and their respective binding patterns to theVEGFRs are shown in FIG. 2. VEGFR-1 and neuropilin-1 (NRP-1) areexpressed in blood vascular ECs, VEGFR-3 and NRP-2 in lymphatic ECs, andVEGFR-2 occurs in both cell lineages. VEGFR-2 is the mainsignal-transducing receptor, as it activates several downstreamsignaling molecules (circles) and induces responses such as cellproliferation, migration, and survival. The protein kinase C(PKC)-mediated MEK/ERK pathway produces proliferation signals, incontrast to activating the PI3-kinase/Akt pathway, which regulates cellsurvival. Focal adhesion kinase (FAK) and PI3-kinase migrate cells bystimulating the reorganization of actin and recruitment ofactin-anchoring proteins to the focal adhesions. VEGF-C and VEGF-D areligands for VEGFR-3, and they can induce LEC survival, migration, andgrowth via activation of the MEK/ERK and PI3-kinase/Akt pathways.However, after proteolytic cleavage, VEGF-C and VEGF-D can also bind andactivate VEGFR-2 and stimulate both BECs and LECs. The distinct butoverlapping receptor specificities and receptor expression patternsdetermine how VEGFs can differentially target both the blood vascularand/or lymphatic endothelium.

The lymphatic system may be assessed through imaging. The imaging maycomprise one or more selected from the group consisting ofcomputer-assisted tomography (CAT), magnetic resonance imaging (MRI),positron emission tomography (PET), lymphoscintigraphy, and radiographyof radiolabeled agents. The imaging may be computer-assisted tomography(CAT). The imaging may be magnetic resonance imaging (MRI). The imagingmay be positron emission tomography (PET). The imaging may belymphoscintigraphy. The imaging may be radiography of radiolabeledagents.

The lymphatic system may be assessed by measuring levels in a tissuesample of one or more first factors selected from the group consistingof D2-40, podoplanin, CD34, and LYVE-1.

The first factor may be D2-40, a monoclonal antibody to an MW 40,000O-linked sialoglycoprotein that reacts with a fixation-resistant epitopeon lymphatic endothelium.

The first factor may be podoplanin.

The first factor may be CD34. Hematopoietic progenitor cell antigenCD34, also known as CD34 antigen, is a protein that in humans is encodedby the CD34 gene. CD34 is a cluster of differentiation in a cell surfaceglycoprotein and functions as a cell-cell adhesion factor. CD34 may alsomediate the attachment of stem cells to bone marrow extracellular matrixor directly to stromal cells.

The first factor may be LYVE-1. Lymphatic vessel endothelial hyaluronanreceptor 1 (LYVE1), also known as extracellular link domain containing 1(XLKD1), is a Link domain-containing hyaladherin, a protein capable ofbinding to hyaluronic acid (HA), homologous to CD44, the main HAreceptor. In humans, it is encoded by the LYVE1 gene.

The tissue sample may be concurrently stained for one or more secondfactors selected from the group consisting of angiopoietin-1,angiopoietin-2, BMP-9, EGF, endoglin, endothelin-1, FGF-1, FGF-2,follistatin, G-CSF, HB-EGF, HGF, IGF, IL-8, leptin, MMP-2, MMP-9, NRP 1,NRP 2, PDGF, PIGF, PLGF, TIE1/2, VEGF-A, VEGF-C, and VEGF-D to determinelevels of these factors within lymphatics.

The second factor may be angiopoietin-1. The second factor may beangiopoietin-2.

The second factor may be BMP-9, also known as GDF2, which contains anN-terminal TGF-beta-like pro-peptide (prodomain) (residues 56-257) and aC-terminal transforming growth factor beta superfamily domain (325-428).GDF2 (BMP9) is secreted as a pro-complex consisting of the BMP9 growthfactor dimer non-covalently bound to two BMP9 prodomain molecules in anopen-armed conformation.

The second factor may be epidermal growth factor (EGF), which stimulatescell growth and differentiation by binding to its receptor, EGFR. HumanEGF is a 6-kDa protein with 53 amino acid residues and threeintramolecular disulfide bonds.

The second factor may be endoglin (ENG), which is a type I membraneglycoprotein on cell surfaces and is part of the TGF beta receptorcomplex. Endoglin is also commonly referred to as CD105, END, FLJ41744,HHT1, ORW, and ORW1. Endoglin has a crucial role in angiogenesis,therefore, making it an important protein for tumor growth, survival,and metastasis of cancer cells to other locations in the body.

The second factor may be endothelin-1 (ET-1), also known aspreproendothelin-1 (PPET1), which is a potent vasoconstrictor that inhumans is encoded by the EDN1 gene and produced by vascular endothelialcells. The protein encoded by this gene is proteolytically processed torelease a secreted peptide termed endothelin 1. Endothelin 1 is one ofthree isoforms of human endothelin.

The second factor may be heparin-binding growth factor 1 (FGF-1) is aprotein that in humans is encoded by the FGF1 gene.

The second factor may be heparin-binding growth factor 2 (FGF-2) is aprotein that in humans is encoded by the FGF2 gene. FGF-1.

The second factor may be follistatin, also known as “activin-bindingprotein.” Follistatin is a protein that in humans is encoded by the FSTgene. Follistatin is an autocrine glycoprotein expressed in all tissuesof higher animals.

The second factor may be a granulocyte-colony stimulating factor (G-CSFor GCSF), also known as colony-stimulating factor 3 (CSF 3). G-CSF is aglycoprotein that stimulates the bone marrow to produce granulocytes andstem cells and release them into the bloodstream. Functionally, it is acytokine and hormone, a type of colony-stimulating factor produced bymany different tissues. The pharmaceutical analogs of naturallyoccurring G-CSF are called filgrastim and lenograstim. G-CSF alsostimulates the survival, proliferation, differentiation, and function ofneutrophil precursors and mature neutrophils.

The second factor may be a heparin-binding EGF-like growth factor(HB-EGF), which is a member of the EGF family of proteins that in humansis encoded by the HBEGF gene. The HB-EGF-like growth factor issynthesized as a membrane-anchored mitogenic and chemotacticglycoprotein. An epidermal growth factor produced by monocytes andmacrophages, due to an affinity for heparin is termed HB-EGF. It plays arole in wound healing, cardiac hypertrophy, and heart development andfunction. HB-EGF is an 87-amino acid glycoprotein that displays highlyregulated gene expression. Ectodomain shedding results in the solublemature form of HB-EGF, which influences the mitogenicity and chemotacticfactors for smooth muscle cells and fibroblasts. The transmembrane formof HB-EGF is the unique receptor for diphtheria toxin and functions injuxtracrine signaling in cells. Both forms of HB-EGF participate innormal physiological processes and in pathological processes, includingtumor progression and metastasis, organ hyperplasia, and atheroscleroticdisease. HB-EGF can bind two locations on cell surfaces: heparan sulfateproteoglycans and EGF-receptor effecting cell to cell interactions.

The second factor may be a hepatocyte growth factor (HGF) or scatterfactor (SF). HGF is a paracrine cellular growth, motility, andmorphogenic factor. HGF is secreted by mesenchymal cells and targets andacts primarily upon epithelial cells and endothelial cells, but alsoacts on hemopoietic progenitor cells and T cells. It has a major role inembryonic organ development, specifically in myogenesis, adult organregeneration, and wound healing.

The second factor may be insulin-like growth factor 1 (IGF-1), alsocalled somatomedin C. IFG-1 is a protein that in humans is encoded bythe IGF1 gene. IGF-1 has also been referred to as a “sulfation factor,”and its effects were termed “nonsuppressible insulin-like activity”(NSILA).

The second factor may be interleukin 8 (IL-8 or chemokine (C-X-C motif)ligand 8, CXCL8) is a chemokine produced by macrophages and other celltypes such as epithelial cells, airway smooth muscle cells, andendothelial cells. Endothelial cells store IL-8 in their storagevesicles, the Weibel-Palade bodies. In humans, the interleukin-8 proteinis encoded by the CXCL8 gene. IL-8 is initially produced as a precursorpeptide of 99 amino acids, which then undergoes cleavage to createseveral active IL-8 isoforms. In culture, a 72-amino acid peptide is amajor form secreted by macrophages.

The second factor may be leptin. Leptin, the “satiety hormone,” is ahormone made by adipose cells that helps to regulate energy balance byinhibiting hunger. Leptin is opposed by the actions of the hormoneghrelin, the “hunger hormone.” Both hormones act on receptors in thearcuate nucleus of the hypothalamus to regulate appetite to achieveenergy homeostasis. In obesity, a decreased sensitivity to leptinoccurs, resulting in an inability to detect satiety despite high energystores.

The second factor may be matrix metalloproteinase 2 (MMP-2). Also knownas 72 kDa type IV collagenase and gelatinase A, MMP-2 is an enzyme thatin humans is encoded by the MMP2 gene. The MMP2 gene is on chromosome 16at position 12.2.

The second factor may be matrix metalloproteinase 9 (MMP-9). Also knownas 92 kDa type IV collagenase, 92 kDa gelatinase, or gelatinase B(GELB), MMP-9 is a matrixin, a class of enzymes that belong to thezinc-metalloproteinases family involved in the degradation of theextracellular matrix. In humans, the MMP9 gene encodes for a signalpeptide, a propeptide, a catalytic domain with inserted three repeats offibronectin type II domain followed by a C-terminal hemopexin-likedomain.

The second factor may be neuropilin-1 (NRP-1) is a protein that inhumans is encoded by the NRP1 gene. In humans, the neuropilin 1 gene isat 10p11.22.

The second factor may be neuropilin-2 (NRP-2). NRP-2 is a protein thatin humans is encoded by the NRP2 gene. This gene encodes a member of theneuropilin family of receptor proteins. The encoded transmembraneprotein binds to SEMA3C protein {sema domain, immunoglobulin domain(Ig), short basic domain, secreted, (semaphorin) 3C} and SEMA3F protein{sema domain, immunoglobulin domain (Ig), short basic domain, secreted,(semaphorin) 3F}, and interacts with vascular endothelial growth factor(VEGF). This protein may play a role in cardiovascular development, axonguidance, and tumorigenesis. Multiple transcript variants encodingdistinct isoforms have been identified for this gene.

The second factor may be a platelet-derived growth factor (PDGF) is oneof many growth factors that regulate cell growth and division. PDGFplays a significant role in blood vessel formation (angiogenesis), thegrowth of blood vessels from already-existing blood vessel tissue,mitogenesis, i.e., proliferation, of mesenchymal cells such asfibroblasts, osteoblasts, tenocytes, vascular smooth muscle cells, andmesenchymal stem cells as well as chemotaxis, the directed migration, ofmesenchymal cells. Platelet-derived growth factor is a dimericglycoprotein that can be composed of two A subunits (PDGF-AA), two Bsubunits (PDGF-BB), or one of each (PDGF-AB).

The second factor may be phosphatidylinositol-glycan biosynthesis classF protein (PIGF).

The second factor may be a placental growth factor (PGF), a protein thatin humans is encoded by the PGF gene. PGF is a member of the VEGF(vascular endothelial growth factor) sub-family. The main source of PGFduring pregnancy is the placental trophoblast. PGF is also expressed inmany other tissues, including the villous trophoblast.

The second factor may be tyrosine kinase with immunoglobulin-like andEGF-like domains 1 and 2 (TIE1/2), which is an angiopoietin receptorwhich in humans is encoded by the TIE1 gene.

The second factor may be vascular endothelial growth factor A (VEGF-A).The second factor may be vascular endothelial growth factor C (VEGF-C).The second factor may be vascular endothelial growth factor D (VEGF-D).

The lymphatic system may be assessed from elevated levels measured usinga flow-cytometry-based multiplex assay or an enzyme-linked immunosorbentassay. The lymphatic system may be assessed from elevated levelsmeasured using a flow-cytometry-based multiplex assay. The lymphaticsystem may be assessed from elevated levels measured using anenzyme-linked immunosorbent assay.

The lymphatic system may be assessed from expression levels measured byone or more techniques selected from the group consisting ofimmunohistochemistry, gene expression profiling, and polymerase chainreaction (PCR)-based cDNA amplification of alymphangiogenesis-regulating gene. The lymphatic system may be assessedfrom expression levels measured by immunohistochemistry. The lymphaticsystem may be assessed from expression levels measured by geneexpression profiling. The lymphatic system may be assessed fromexpression levels measured by polymerase chain reaction (PCR)-based cDNAamplification of a lymphangiogenesis-regulating gene.

The lymphangiogenesis-regulating gene may be one or more selected fromthe group selected from angiopoietin-1, angiopoietin-2, BMP-9, EGF,endoglin, endothelin-1, FGF-1, FGF-2, follistatin, G-CSF, HB-EGF, HGF,IGF, IL-8, leptin, MMP-2, MMP-9, NRP 1, NRP 2, PDGF, PIGF, PLGF, TIE1/2,VEGF-A, VEGF-C, and VEGF-D. The lymphangiogenesis-regulating gene may beangiopoietin-1. The lymphangiogenesis-regulating gene may beangiopoietin-2. The lymphangiogenesis-regulating gene may be BMP-9. Thelymphangiogenesis-regulating gene may be EGF. Thelymphangiogenesis-regulating gene may be endoglin. Thelymphangiogenesis-regulating gene may be endothelin-1. Thelymphangiogenesis-regulating gene may be FGF-1. Thelymphangiogenesis-regulating gene may be FGF-2. Thelymphangiogenesis-regulating gene may be follistatin. Thelymphangiogenesis-regulating gene may be G-CSF. Thelymphangiogenesis-regulating gene may be HB-EGF. Thelymphangiogenesis-regulating gene may be HGF. Thelymphangiogenesis-regulating gene may be IGF. Thelymphangiogenesis-regulating gene may be IL-8. Thelymphangiogenesis-regulating gene may be leptin. Thelymphangiogenesis-regulating gene may be MMP-2. Thelymphangiogenesis-regulating gene may be MMP-9. Thelymphangiogenesis-regulating gene may be NRP 1. Thelymphangiogenesis-regulating gene may be NRP 2. Thelymphangiogenesis-regulating gene may be PDGF. Thelymphangiogenesis-regulating gene may be PIGF. Thelymphangiogenesis-regulating gene may be PLGF. Thelymphangiogenesis-regulating gene may be TIE1/2. Thelymphangiogenesis-regulating gene may be VEGF-A. Thelymphangiogenesis-regulating gene may be VEGF-C. Thelymphangiogenesis-regulating gene may be VEGF-D.

The lymphatic system may be assessed by profiling immune cells directlyin a specimen by flow cytometry, mass spectrometry, cell labeling, orany combination thereof. The lymphatic system may be assessed byprofiling immune cells directly in a specimen by flow cytometry. Thelymphatic system may be assessed by profiling immune cells directly in aspecimen by mass spectrometry. The lymphatic system may be assessed byprofiling immune cells directly in a specimen cell labeling.

The lymphatic system may be assessed by measuring one or more markersselected from the group selected from angiopoietin-1, angiopoietin-2,heparin-binding factor midkine, BMP-9, EGF, endoglin, endothelin-1,FGF-1, FGF-2, follistatin, G-CSF, HB-EGF, HGF, IGF, IL-8, leptin, MMP-2,MMP-9, NRP 1, NRP 2, PDGF, PIGF, PLGF, TIE1/2, VEGF-A, VEGF-C, andVEGF-D. The marker may be angiopoietin-1. The marker may beangiopoietin-2. The marker may be heparin-binding factor midkine. Themarker may be BMP-9. The marker may be EGF. The marker may be endoglin.The marker may be endothelin-1. The marker may be FGF-1. The marker maybe FGF-2. The marker may be follistatin. The marker may be G-CSF. Themarker may be HB-EGF. The marker may be HGF. The marker may be IGF. Themarker may be IL-8. The marker may be leptin. The marker may be MMP-2.The marker may be MMP-9. The marker may be NRP 1. The marker may be NRP2. The marker may be PDGF. The marker may be PIGF. The marker may bePLGF. The marker may be TIE/2. The marker may be VEGF-A. The marker maybe VEGF-C. The marker may be VEGF-D.

The art teaches that a patient's response to immune therapy depends onthe PD-1 or PD-Li expression or tumor neoantigen status. For example,hypermutant, microsatellite instability-high, DNA mismatch repairdeficient (dMMR), or high neoantigen burden phenotype tumors respondstrongly to checkpoint immune therapies. The cancers which are low in ordo not express PD-1 or PDL-1 or are not hypermutant, not dMMR,microsatellite instability-low or normal, or have low neoantigenburdens, do not respond to checkpoint immune therapy. Breast cancer,particularly triple-negative (iER2−, ER−, PR−), ER+/HER2 negative, andinflammatory breast cancers, microsatellite instability low or normal,nonhypermutant/DNA mismatch repair low or normal colorectal cancers, andglioblastomas multiforme (GBMs), pancreatic cancer, sarcomas, andprostate cancers do not respond well to immune-modulating therapies. Onthe contrary, the present disclosure shows that the tumor typesdescribed above respond to immune checkpoint inhibition when treated inrelation to lymphatic dysfunction, independent of PD-1/PD-L1,microsatellite instability degree, dMMR status, orneoantigen/hypermutant tumor status or type (see Example 4).

II. Methods for Treating Cancer

Also provided herein is a method for treating cancer in a subject inneed thereof. The method comprises, when a lymphatic system in a subjectis dysregulated, administering to the subject a therapeutic amount of adrug to regulate the dysregulated lymphatic system, and administering tothe subject a therapeutic amount of an immune modifying therapy.Alternatively, when the lymphatic system is dysregulated, a therapeuticamount of an immune-modulating therapy is administered to the subject,which immune-modulating therapy operates independently of immune-cellpriming, antigen trafficking, antigen presentation, and any combinationthereof.

The art suggests that tumors with high levels of lymphangiogenesisshould have a better prognosis and better response to immune therapiesbecause they have higher tumor immune cell infiltrates. To the contrary,following the present disclosure, such tumors should be treated withboth immune modulation therapies, including immune checkpointinhibitors, and a modulator of lymphatic biology, such as a VEGR-3inhibitor, VEGF-C, VEGF-D, NRP 1, NRP 2, or CCPE1.

As such, therapies that modulate (stimulate or inhibit) immune biologyat or downstream of this step are not effective monotherapies. They mustbe replaced with alternate therapies not dependent on these steps ormechanisms and treated with alternate therapies, or theseimmune-modulating therapies will either need to be independent of immunecell priming, antigen priming, or presentation or the immune-modulatingtherapies that are dependent on these steps will need to be augmented orchanged by agents that help to limit or overcome these issues. Further,cancer patients treated with immune-modulating therapies dependent onimmune cell priming, antigen trafficking, or antigen presentation (e.g.,immune checkpoint therapies) or patients that have dysregulated,dysfunctional or perturbed lymphatic systems can as a class all beaugmented, and their clinical profiles improved through augmentationwith such agents (antibody or antibody derivatives, or small molecularor small molecule derivatives) that regulate lymphatic angiogenesis.

Further, based on the evaluation of the status of the lymphatic systemin the cancer subject, the potential treatment with an immune-modulatingtherapy is assessed. If subjects are determined to have dysregulation oftheir lymphatic system by having abnormal lymphatic system features,then the treatment with any immune-modulating therapy that depends onefficient immune cell priming or antigen presentation is either aborted,deferred, or is augmented by a treatment method that modulates thelymphatic system to overcome or offset the dysfunctions.

Targeting lymphatics surrounding cancer therapy exclusively focuses onlymphatics as conduits for metastasis and as a means of limitingmetastasis by preventing cancer cell spread along these conduits. Theprior art focuses on these therapies in the context of providing moresupport around tumor-associated blood vessel angiogenesis by coveringthe vascular angiogenesis pathway that existing and marketed do notcover. The art does not teach specific and highly selective inhibitorsof lymphangiogenesis for augmenting or aiding immune modulation orimmune therapies. The prior art also does not teach aiding immunecheckpoint inhibitors as a principal means of augmenting the effects ofthese therapies, supporting or boosting immune therapies. Additionally,the prior art does not teach lymphangiogenesis inhibitors in combinationwith immune-modulating therapies in patients with lymphaticdysregulation, dysfunction, or perturbation.

The prior art does not teach treating patients with dysfunctional,dysregulated, or perturbed lymphatic systems characterized by lymphaticinvasion and or lymphangitic carcinomatosis. While the prior artsuggested anti-lymphangiogenic agents in ongoing clinical trials, theagents were selected solely for their known role as primary cancertherapies that target and inhibit vascular angiogenesis and do notselectively inhibit lymphangiogenesis. They are nonspecific agents withhigh general specificity for the entire VEGF family of receptors andmany other angiogenesis-related targets (e.g., PDGF-BB, HGF, etc.), notspecific and selective agents for VEGF-C/D and VEGFR 3. Additionally,the prior art does not teach lymphatic biology specific mediators fortreating dysregulated lymphatics in the singular role of cancerimmunotherapy for targeting lymphatic dysregulation so that immunetherapies can more effectively function.

In a clinical setting, a major challenge in the treatment of establishedmetastatic disease after the primary tumor has been surgically removed,eradicated by other means, or is unresectable. Following the presentdisclosure, established metastatic disease by blocking lymphangiogenesisusing antagonists of VEGF-C receptors, VEGFR-3 and VEGFR-2 incombination with cancer immune-modulating therapies which can includebut are not limited to immune checkpoint inhibitors and in the settingof a dysregulated lymphatic system, or tumor-associated lymphaticinvasion, lymphangitic carcinomatosis, or impaired antigen presentation,immune cell activation or priming alone or due to an impaired ordysregulated, dysfunctional or perturbed lymphatic system.

Specifically, the present disclosure provides a method for inhibitingestablished tumor metastasis in a subject comprising administering tosaid subject a therapeutically effective amount of one or more VEGFR-3antagonist(s) and optionally one or more VEGFR-2 antagonist(s) withcancer immune-modulating therapies which can include but are not limitedto immune checkpoint inhibitors and in the setting of a dysregulatedlymphatic system, or tumor-associated lymphatic invasion, lymphangiticcarcinomatosis, or impaired antigen presentation, immune cell activationor priming alone or due to an impaired or dysregulated, dysfunctional orperturbed lymphatic system. A method is provided for inhibitinglymphangiogenesis in a subject with a metastatic disease comprisingadministering to said subject a therapeutically effective amount of oneor more VEGFR-3 antagonist(s) and optionally one or more VEGFR-2antagonist(s) in combination with cancer immune-modulating therapieswhich can include but are not limited to immune checkpoint inhibitorsand in the setting of a dysregulated lymphatic system, ortumor-associated lymphatic invasion, lymphangitic carcinomatosis, orimpaired antigen presentation, immune cell activation or priming aloneor due to an impaired or dysregulated, dysfunctional or perturbedlymphatic system.

A. Lymphatic System

The lymphatic system comprises capillaries and larger collecting vesselscontinuously lined by endothelial cells, which return extravasated fluidand macromolecules from the interstitial space back to the bloodcirculation. Thus, the lymphatic system plays a vital role in theregulation of fluid, protein, and pressure equilibrium in tissues. Bydirecting leukocytes and antigens from tissues to the lymph nodes,lymphatic vessels also have a key function in immune surveillance.Dysfunction of the lymphatic system results in lymphedema, a chronic anddisabling condition for no treatments now available. Breast cancertreatment is associated with lymphedema, which often develops followingsurgical removal of lymph nodes and radiation therapy.

The lung is a common site for metastasis of many tumors, includingcommon tumors such as breast, colorectal, prostate, bronchial,head-and-neck, and renal cancers. Pulmonary nodules are the most commonmanifestation of metastatic cancer in the lungs. Without wishing to bebound by theory, they are thought to be derived from tumor emboli whicharrest in the lung capillaries and invade into the surrounding lungtissue. The involvement of pulmonary lymphatic vessels with cancer isless diagnosed because of imaging difficulties. At necropsy, metastasesvia pulmonary lymphatics and bronchial arteries are often seen.

Involving lung lymphatics with cancer is a hallmark of a very aggressivemetastatic disease, designated “lymphangitic carcinomatosis.” Theprognosis for a patient with this clinical picture is extremely poor;50% of the patients die within 3 months of diagnosis. Althoughlymphangitic spread can be caused by any malignant cancer, it mostcommonly results from tumors originating in the breast, stomach,pancreas, lung, or prostate. This phenomenon is also caused by primarypulmonary carcinoma, especially small cell carcinoma and adenocarcinoma.Because of the extremely aggressive nature of this disease, there is agreat need for early diagnosis and treatment. Before the presentdisclosure, no treatment improved the outcome of patients withlymphangitic carcinomatosis.

Lymphangitic carcinomatosis is an aggressive disease that has been seenin association with many common metastatic cancers such as breast,gastric, pancreatic, prostate cancer, and others. Primary lung cancercan also present in the form of lymphangitic carcinomatosis, suggestingthat targeting of VEGF-C/VEGFR-3 in lung cancer could be a treatmentoption for slowing the progression of lung cancer in combination withcancer immune-modulating therapies which can include, but are notlimited, to immune checkpoint inhibitors and in the setting of adysregulated lymphatic system, or tumor-associated lymphatic invasion,lymphangitic carcinomatosis, or impaired antigen presentation, immunecell activation or priming alone or due to an impaired or dysregulated,dysfunctional or perturbed lymphatic system.

Clinically, lymphangitic carcinomatosis is characterized by malignantcells in the lymphatic vessels localized in the peri-bronchovasculararea, in the interlobular septa, and the centrilobular region.Associated pleural involvement is common. Edema resulting from blockageof lymphatic drainage and a desmoplastic reaction is common and cancontribute to interstitial thickening. Hilar and mediastinallymphadenopathy is present in 20-40% of patients, and pleural effusionsare present in 30-50% of patients.

The dysregulated lymphatic system may be characterized by one or moreselected from the group consisting of abnormal lymphatic development,lymphatic proliferation, lymphangiogenesis, impaired lymphatic vesselfunction, dysregulated lymphatic vessel function, augmented tumor celllymphatic infiltration, lymphangitic carcinomatosis, abnormalfunctioning or homeostatic regulation, lymphatic remodeling, physicalpressure upon lymphatics, altered tumoral lymphatic development, alteredtumoral lymphangiogenesis, and output blockage of lymphatic structuresin lymphatic organs. The dysregulated lymphatic system may becharacterized by abnormal lymphatic development. The dysregulatedlymphatic system may be characterized by lymphatic proliferation. Thedysregulated lymphatic system may be characterized by lymphangiogenesis.The dysregulated lymphatic system may be characterized by impairedlymphatic vessel function. The dysregulated lymphatic system may becharacterized by dysregulated lymphatic vessel function. Thedysregulated lymphatic system may be characterized by augmented tumorcell lymphatic infiltration. The dysregulated lymphatic system may becharacterized by lymphangitic carcinomatosis. The dysregulated lymphaticsystem may be characterized by abnormal functioning or homeostaticregulation. The dysregulated lymphatic system may be characterized bylymphatic remodeling. The dysregulated lymphatic system may becharacterized by physical pressure upon lymphatics. The dysregulatedlymphatic system may be characterized by altered tumoral lymphaticdevelopment. The dysregulated lymphatic system may be characterized byaltered tumoral lymphangiogenesis. The dysregulated lymphatic system maybe characterized by the output blockage of lymphatic structures inlymphatic organs.

B. Drug to Regulate the Lymphatic System

The drug to regulate the lymphatic system may be administered before thetherapeutic amount of the immune modifying therapy.

The drug to regulate the lymphatic system may be administeredconcurrently with the therapeutic amount of the immune modifyingtherapy.

The drug to regulate the lymphatic system may be an inhibitor or anantagonist for a target selected from the group consisting of (1)inhibitors and antagonists of VEGFR-2, heparin-binding factor midkine,VEGFR-3, VEGF-C, VEGF-D, Ang2/Tie2, NRP 1, NRP 2, CCPE1, CSF1, CSFR1,and CCL21; (2) a regulator of lymphatic endothelial cell metabolism; (3)an enzyme involved in lymphatic endothelial cell fatty acid oxidation;(4) a regulator of PROX1; and any combination thereof.

The drug to regulate the lymphatic system may inhibit VEGFR-2. The drugto regulate the lymphatic system may antagonize VEGFR-2. The drug toregulate the lymphatic system may inhibit VEGFR-3. The drug to regulatethe lymphatic system may antagonize VEGFR-3. The drug to regulate thelymphatic system may inhibit VEGF-C. The drug to regulate the lymphaticsystem may antagonize VEGF-C. The drug to regulate the lymphatic systemmay inhibit VEGF-D. The drug to regulate the lymphatic system mayantagonize VEGF-D. The drug to regulate the lymphatic system may inhibitAng2/Tie2. The drug to regulate the lymphatic system may antagonizeAng2/Tie2. The drug to regulate the lymphatic system may inhibit NRP 1.The drug to regulate the lymphatic system may antagonize NRP 1. The drugto regulate the lymphatic system may inhibit NRP 2. The drug to regulatethe lymphatic system may antagonize NRP 2. The drug to regulate thelymphatic system may inhibit a CCPE1. The drug to antagonize thelymphatic system may be a CCPE1.

The drug to regulate the lymphatic system may inhibit colony-stimulatingfactor 1 (CSF1). The drug to regulate the lymphatic system mayantagonize CSF1. Also known as macrophage colony-stimulating factor(M-CSF) is a secreted cytokine that influences hematopoietic stem cellsto differentiate into macrophages or other related cell types.Eukaryotic cells also produce M-CSF to combat intercellular viralinfection. It is an experimentally described colony-stimulating factor.M-CSF binds to the colony-stimulating factor 1 receptor. It may also beinvolved in placental development.

The drug to regulate the lymphatic system may inhibit thecolony-stimulating factor 1 receptor (CSFR1). The drug to regulate thelymphatic system may antagonize CSFR1. Also known as macrophagecolony-stimulating factor receptor (M-CSFR) and CD115 (Cluster ofDifferentiation 115), this target is a cell-surface protein encoded, inhumans, by the CSF1R gene (also known as c-FMS). It is a receptor for acytokine called colony-stimulating factor 1.

The drug to regulate the lymphatic system may inhibit Chemokine (C-Cmotif) ligand 21 (CCL21). The drug to regulate the lymphatic system mayantagonize CCL21. CCL21 is a small cytokine belonging to the CCchemokine family. This chemokine is also known as 6Ckine (because it hassix conserved cysteine residues instead of the four cysteines typical tochemokines), exodus-2, and secondary lymphoid tissue chemokine (SLC).The gene for CCL21 is on human chromosome 9. CCL21 elicits its effectsby binding to a cell surface chemokine receptor known as CCR7.

The drug to regulate the lymphatic system may inhibit a regulator oflymphatic endothelial cell metabolism. The drug to regulate thelymphatic system may antagonize a regulator of lymphatic endothelialcell metabolism. The drug to regulate the lymphatic system may inhibitan enzyme involved in lymphatic endothelial cell fatty acid oxidation.The drug to regulate the lymphatic system may antagonize an enzymeinvolved in lymphatic endothelial cell fatty acid oxidation.Non-limiting examples of drugs that inhibit or antagonize fatty acidoxidation include a 3-KAT inhibitor, such as trimetazidine andranolazine; a CPT1 inhibitor, such as etomoxir, perhexiline, andoxfenicine; and a mitochondrial thiolase inhibitor, such as4-bromocrotonic acid.

For example, the regulator of lymphatic endothelial cell metabolism maybe a member of the carnitine palmitoyltransferase I (CPT1) enzymefamily. Also known as carnitine acyltransferase I, CPTI, CAT1,CoA:carnitine acyltransferase (CCAT), or palmitoyl-CoA transferase I,this target is a mitochondrial enzyme responsible for the formation ofacylcarnitines by catalyzing the transfer of the acyl group of along-chain fatty acyl-CoA from coenzyme A to 1-carnitine. The product isoften palmitoylcarnitine, but other fatty acids may be substrates.Isoforms of CPT1 include CPT1A, CPT1B, and CPT1C. CPT1 is associatedwith the outer mitochondrial membrane. This enzyme can be inhibited bymalonyl CoA, the first committed intermediate produced during fatty acidsynthesis. Its role in fatty acid metabolism makes CPT1 important inmany metabolic disorders, such as diabetes. Since its crystal structureis not known, its exact mechanism of action remains to be determined.

The drug to regulate the lymphatic system may inhibit the regulator ofProspero homeobox protein 1 (PROX1). The drug to regulate the lymphaticsystem may antagonize the regulator of PROX1. PROX1 is a protein that inhumans is encoded by the PROX1 gene. PROX1 is produced primarily in thedentate gyrus in the mouse and in the dentate gyrus and white matter inhumans.

A combination of drugs may regulate the lymphatic system. The drug toregulate the lymphatic system may comprise a VEGFR-2 inhibitor and aVEGFR-3 inhibitor.

A member of the vascular endothelial growth factor (VEGF) family,VEGF-C, has been shown as a growth factor for lymphatic vessels. VEGF-Cis a ligand for the receptor tyrosine kinase VEGFR-3, which is expressedon lymphatic endothelial cells. VEGF-C also binds to and activatesVEGFR-2, which is expressed by lymphatic and by blood endothelium and isalso used by VEGF-A, a major angiogenesis factor. In tumors, VEGFR-3 isexpressed by lymphatic endothelial cells and by the subset of bloodvessels, but not by tumor cells. The important role of VEGF-C andVEGFR-3 signaling in developmental and postnatal lymphangiogenesis hasbeen documented. Several studies have also shown that VEGF-C/VEGFR-3signaling aids the spread of metastases from the primary tumor into thelymph nodes.

Several studies have also shown that an increase in lymph nodemetastases in mice bearing VEGF-C-expressing primary tumors correlatesto an increase in distant metastases. VEGF-C increased tumorlymphangiogenesis, and cancer spread to the lymph nodes, which wasassociated with an increased metastatic burden in the lung inexperimental models of breast cancer, prostate cancer, and melanoma.Conversely, studies in mouse models of breast cancer, prostate cancer,and melanoma have shown that blocking VEGF-C/VEGFR-3 inhibits tumorlymphangiogenesis and prevents lymph node metastasis in the presence ofa primary tumor, and so reduces the risk of distant metastasis. Based onthese findings, VEGF-C/VEGFR-3-mediated lymphangiogenesis would not beconsidered a target for cancer treatment after the removal of theprimary tumor.

Non-limiting examples of useful VEGFR-3 antagonists include antagonistantibodies and fragments thereof, soluble polypeptides that inhibit theactivity of VEGFR-3 or VEGFR-2 (e.g., an extracellular domain of aVEGFR-3 or VEGFR-2 protein or a derivative thereof), small moleculeinhibitors (e.g., small molecule inhibitors of kinases and/or signalingpathways relevant for VEGFR-3 and/or VEGFR-2 signal transduction), andinhibitors of VEGFR-3 and/or VEGFR-2 expression (e.g., siRNAs, shRNAs,antisense oligonucleotides, ribozymes, etc.). The VEGFR-3 antagonist maybe an anti-VEGFR-3 antibody or an antigen-binding part thereof. SuchVEGFR-3 antagonists may be the monoclonal antibodies mR4-31C1, orVGX-100, or IMC-3C5, OPT-302, or molecules or compounds with similar orderivatives structures or small molecules with the same, similar orderivative structures as SAR-131675.

The VEGFR-2 antagonist may be an anti-VEGFR-2 antibody or anantigen-binding portion thereof. In one specific embodiment, such aVEGFR-2 antagonist is the monoclonal antibody DC101. Such anti-VEGFR-2antibody or the anti-VEGFR-3 antibody may bind an extracellular domainof VEGFR-2 or VEGFR-3, respectively, and can block the interaction ofVEGF-C, VEGF-D, and/or VEGF-A with VEGFR-2 or VEGFR-3. In oneembodiment, the antibody is capable of binding to its target (i.e.,VEGFR-2 or VEGFR-3) with an affinity of at least about 1×10⁷⁻⁶ M, of atleast about 1×10⁻⁷ M, of at least about 1×10⁻⁸ M, or of at least about1×10⁻⁹M.

The anti-VEGFR-2 antibody or the anti-VEGFR-3 antibody may be, e.g., achimeric antibody, a primatized antibody, a humanized antibody, or anantigen-binding portion thereof. Humanized antibodies may include one ormore CDR from the monoclonal antibody DC101 or one or more CDR from themonoclonal antibody mR4-31C1.

The antigen-binding part of the antibody can be, e.g., an F(ab′) 2, aFab, an Fv, an scr′v, or a single domain antibody.

The VEGFR-2 antagonist or the VEGFR-3 antagonist may be a solublepolypeptide antagonist. Such soluble polypeptide antagonist comprises anextracellular domain of a VEGFR-2 protein or an extracellular domain ofa VEGFR-3 protein or an amino acid sequence that is at least 90%, atleast 95%, at least 97%, or at least 99% identical to the extracellulardomain of a VEGFR-2 protein or a VEGFR-3 protein. Optionally, one ormore soluble peptide antagonists can further comprise apost-translational modification. Non-limiting examples of suchpost-translational modifications include, e.g., acetylation,carboxylation, glycosylation, phosphorylation, lipidation, acylation,the addition of a non-amino acid element (such as, e.g., polyethyleneglycol, a lipid, a poly- or mono-saccharide, or a phosphate), andaddition of a fusion domain (such as, e.g., polyhistidine, Glu-Glu,glutathione S transferase (GST), thioredoxin, protein A, protein G, animmunoglobulin heavy chain constant region (Fc), a maltose-bindingprotein (MBP), green fluorescent protein (GFP), or an epitope tag).Fusion domains can further comprise a protease cleavage site (such ase.g., FactorXa or Thrombin).

The pattern of metastatic spread to the lungs seen with VEGF-Cexpressing cells in an MDA-MB-435/VEGF-C mouse model for breast cancerclosely resembles Lymphangitic Carcinomatosis aggressive metastaticphenotype in human cancer patients. As described in greater detailbelow, tumors that do not express VEGF-C do not show any evidence oflymphatic involvement in the lungs, while VEGF-C helps lunglymphangiogenesis, tumor cell entry into the lung lymphatics and growthwithin, creating a niche for tumor expansion within the lung as well asa route for dissemination to the thoracic lymph nodes. Thus, VEGF-Cexpression by tumor cells drastically changes the pattern of metastaticdisease and aids disease progression.

The VEGF-C/VEGFR-3 pathway and the lymphatic vessels are targets fortreating established metastatic disease with cancer immune-modulatingtherapies which can include, but are not limited, to immune checkpointinhibitors and in the setting of a dysregulated lymphatic system, ortumor-associated lymphatic invasion, lymphangitic carcinomatosis, orimpaired antigen presentation, immune cell activation or priming aloneor due to an impaired or dysregulated, dysfunctional or perturbedlymphatic system.

Systemic treatment with VEGFR-3 antagonistic antibodies (mR4-31C1,ImClone Systems, a subsidiary of Eli Lilly and Company, Indianapolis,Ind.) suppressed tumor lymphangiogenesis and inhibited lymph nodemetastasis of MDA-MB-435 cells expressing high levels of VEGF-C(MDA/VEGF-C). Furthermore, combination therapy with a modulator oflymphatic biology, including but not limited to anti-VEGFR-3 antibodieswith a cancer immune-modulating therapy including, but not limited to,an immune checkpoint inhibitor, is more potent in decreasing metastasesthan treatment with either antibody or therapy alone. The effects ofcombination treatment were studied in an intervention regimen, in whichthe treatment began when tumors and metastases were established, fourweeks after the orthotopic tumor cell inoculation into the mammary fatpads. Joint treatment with the antagonistic antibodies to VEGFR-2 andVEGFR-3 also significantly decreased lung metastases.

To understand the mechanism by which combined treatment inhibitsmetastasis, its effects on the primary tumor were investigated. Jointtreatment did not result in greater inhibition of primary tumor growththan treatment with the anti-VEGFR-2 antibody alone. (Blocking VEGFR-3had no effect on primary tumor growth.) Analysis of tumor vasculatureshowed that double-treatment was also not more potent in inhibitingtumor lymphangiogenesis or angiogenesis, as compared to single antibodytreatments. These data demonstrated that the effects of combinedtreatment on metastases could not be explained by changes in the tumorvasculature or growth of the primary tumor.

Events downstream from the primary tumor, i.e., in the lymph nodes, maybe important for the observed inhibition of metastases with the jointtreatment. The pattern of lymphatic and blood vasculature intumor-draining lymph nodes of control and treated mice was examined byimmunofluorescent staining using LYVE-1 and CD34 antibodies,respectively. The results showed that MDA-MB-435/VEGF-C tumors inducedprominent lymphangiogenesis in tumor-draining axillary lymph nodes.LYVE-1 lymphatic vessels were restricted to the medullary zone andsubcapsular sinuses, while no LYVE-1 structures were seen in the lymphnode cortex. Tumor draining lymph nodes increased in size when comparedto control lymph nodes of normal mice.

Blocking VEGFR-3 reduced the lymphatic vessel area and lymph node sizein the tumor-draining lymph nodes to a moderate extent. Blocking VEGFR-2showed moderate inhibition of lymphangiogenesis and a more prominentinhibition of lymph node size. Joint blocking of VEGFR-3 and VEGFR-2drastically inhibited lymph node lymphangiogenesis and dramaticallyreduced lymph node size.

Taken together, analysis of tumor-draining lymph nodes revealed thatjoint blocking of VEGFR-3 and VEGFR-2 was most effective in inhibitinglymph node lymphangiogenesis and lymph node size, as compared to singleantibody treatments. These data showed the importance of concurrentVEGFR-2 and VEGFR-3 signaling for lymph node lymphangiogenesis andstrongly indicated an important role of lymph node lymphangiogenesis forboth lymph node and distant metastases.

The effects of antagonistic antibodies to VEGFR-2 and VEGFR-3 ontumor-induced lymph node angiogenesis were examined. Comparison of theCD34+ vessel pattern in normal and tumor-draining lymph nodes showed anincrease in the density of blood microvasculature within the lymph nodecortex, while the density of blood microvasculature was not altered.Tumors induced angiogenesis in tumor-draining lymph nodes by 80%.Interestingly, the increase in the total number of blood vessels wasdirectly correlated to the increase of lymph node size associated withthe tumor, showing that the blood vessel density per lymph node areastayed unchanged.

Blocking VEGFR-3 alone did reduce the lymph node blood vessel density.Blocking VEGFR-2 drastically reduced blood vessel density (50%). Jointblocking of VEGFR-2 and VEGFR-3 reduced blood vessel numbers slightlymore (64%). Thus, the combination treatment, with antagonisticantibodies to both VEGFR-2 and VEGFR-3, is most effective for theinhibition of lymph node lymphangiogenesis and lymph node size.

VEGF-C expression by tumor cells potently increased the metastaticburden in the lungs. To further understand the mechanism by which VEGF-Cand its receptors aid the formation of distant metastases, the phenotypeof lung metastases formed by MDA-MB-435 and MDA-MB-435/VEGF-C cells wereexamined. These cells were injected orthotopically into the secondmammary fat pads of nude mice, and tumors and metastases could developfor 12 weeks. Tumor size reached an average volume of about 1 cm afterthe 12-week period. At the end of the experiment (12 weeks), 8 out of 8mice (100%) bearing MDA-MB-435 cells or MDA-MB-435/VEGF-C cells had apositive signal in the lungs.

Histopathological analysis of metastases revealed a distinct pattern ofpulmonary metastases by tumor cells expressing high levels of VEGF-C.MDA MB-435/VEGF-C cells showed a unique distribution in the lung ascompared to the non-VEGF-C expressing cells. Metastases fromMDA-MB-435/VEGF-C cells presented as large lesions associated with thebronchi and large pulmonary vessels. In contrast, metastases ofMDA-MB-435 control cells presented as small pulmonary nodules thatlocalized in the lung parenchyma and were not associated with thebronchi. Smooth muscle O-actin staining of the large pulmonary vesselsand airways further showed that metastases from MDA-MB-435 cells had noaffiliation with the large pulmonary vasculature and were often distantfrom large vessels, while VEGF-C overexpressing lesions were often seenin intravascular emboli, localizing in pulmonary arteries. These resultsshow that increased VEGF-C production by metastatic cells resulted in anincrease in lung metastasis and drives a phenotype in which tumor cellsare often seen as endovascular nodules in the peribronchovascularregion.

Lymphatic vessels in lungs infiltrated with MDA/pcDNA tumor cells weredetected in their normal anatomical location, i.e., surrounding bronchi,large pulmonary vessels, and in the pleura, and they were not altered intheir appearance compared to normal lungs not involved with cancer.There was no lymphangiogenesis associated with MDA/pcDNA nodules, andonly seldom were lymphatics seen near these nodules (number ofmetastatic foci with lymphatics present within 200 pum:MDA/pcDNA 6/48;13% vs. MDA/VEGF-C 37/39; 95%). In contrast, VEGF-C-overexpressingmetastatic lesions had pronounced lymphangiogenesis, and lymphaticvessels were distended throughout the lungs with MDA/VEGF-C metastases.Lymphatic vessel area associated with MDA/VEGF-C metastatic foci was, onaverage, 75-fold greater than lymphatic vessel area associated withMDA/pcDNA foci, which had lymphatics in the proximity. The expansion ofthe lymphatic network paralleled an increase in the size of MDA/VEGF-Cmetastases.

Because most VEGF-C overexpressing metastases were found next tobronchi, the relationship between MDA/VEGF-C metastases and the deeplymphatic plexus associated with the bronchial tree was investigated.Lymphatic vessels were detected by using a combination of anti-LYVE-1,anti-podoplanin, and anti-VEGFR-3 antibodies. Strikingly, the bulk ofMDA/VEGF-C metastases seen in the peribronchial area was seen inside ofthe distended lymphatic vessels (31/55 metastatic foci showed lymphaticvessel involvement; 56%). Lymphatic vessels near pulmonary veins werealso massively infiltrated with tumor cells. Furthermore, MDA/VEGF-Cmetastases were often detected in the pleura (MDA/VEGF-C in 5/7 mice;MDA/pcDNA in 1/6 mice), which is another area of lung tissue rich inlymphatics. In sharp contrast, MDA/pcDNA metastases were rarely seenintravascular or even near the lymphatic vasculature. These data showthat VEGF-C aid intralymphatic spread of metastases in the lung.

To investigate whether VEGF-C promotes secondary metastaticdissemination, within the lymphatic network in the lung, lung-draininglymph nodes were analyzed for metastases. At necropsy, mediastinal andhilar lymph nodes from mice bearing VEGF-C overexpressing and controlMDA-MB-435 tumors were evaluated using stereomicroscopy to detect theGFP fluorescence. The incidence of lymph nodes positive for metastasesfrom VEGF-C overexpressing tumor-bearing mice was significantly higher(13/20, 65%) than in mice bearing control tumors (3/20, 15%). To confirmthe presence of tumor cells and to analyze the specific area of themetastases within the mediastinal or hilar lymph nodes, thehistopathology of lymph nodes from mice bearing the VEGF-Cover-expressing tumors was studied. Using podoplanin as a lymphaticvessel marker, metastases were seen in the subcapsular sinus region.These data showed that VEGF-C promotes the lymphatic spread ofmetastases within the lung, promoting secondary tumor spread to thethoracic lymph nodes.

The finding that VEGF-C induces aggressive lymphangitic carcinomatosisphenotype in a mouse breast cancer model suggested that VEGF-C and itsreceptor VEGFR-3 and/or VEGFR-2 could be targets for treating thisdisease. Because in most cancer patients with cancer primary tumor isremoved (unless it is unresectable), an experiment was designed in whichVEGFR-3 signaling was inhibited with antagonistic antibodies after theremoval of the primary tumor. VEGF-C overexpressing MDA-MB-435 cellswere orthotopically injected into the second mammary fat pad of nu/numice, and tumor and lung metastases could develop for 8 weeks. Theextent of metastases was monitored and quantified by in vivobioluminescent imaging. In the 8th week, the primary tumor wassurgically removed, and function-blocking antibodies to VEGFR-3(mP4-31C1, ImClone Systems) were administered at 800 pg/mouse everysecond day, and metastases were analyzed after six weeks of treatment.

A stark contrast between the metastatic patterns between the control andthe mP4-31C1 treated groups was seen, as assessed by histopathologicalanalysis of lung sections. Control samples (MDA/VEGF-C cells) showedmany intravascular metastases seen in the pulmonary arteries associatedwith the bronchial tree and in the lymphatic vessels of theperibronchovascular region, around the pulmonary veins, and in thepleura. Conversely, in lung sections from the mice treated withanti-VEGFR-3 antibodies, metastases were seen primarily in the lungparenchyma and capillaries of the lungs and were less often associatedwith lymphatics, pulmonary arteries, or the bronchial tree. Metastasesassociated with the airways were not found in the lymphatic vessels orin the pulmonary arteries, and there was no lymphangiogenesis. Thetypical phenotype of VEGF-C expressing metastases grow in the lymphaticvessels and present as pulmonary artery tumor emboli. Collectively,these data showed that VEGF-C/VEGFR-3 signaling has a significant rolein driving breast adenocarcinoma metastases towards the clinicalmanifestation of lymphangitic carcinomatosis and that inhibition ofVEGFR-3 can reverse this aggressive phenotype.

C. Immune-Modulating Therapy

The immune-modulating therapy may be selected from the group consistingof an antagonist for immune checkpoint inhibition, an agonist for immuneco-stimulation signal, a stimulatory factor affecting immune cellpriming and activation, a chemotactic agent, cytokine-related immunemodulator, chemotherapeutic immune stimulation, radiotherapeutic immunestimulation, a vaccine, activation of an adaptive immune response, andactivation of innate immune response. Immune-modulating therapy thatworks independent of immune cell priming and antigen presentationinclude adoptive cell transfer and immune cell modification strategies,such as chimeric antigen receptor T cell (CAR-T) therapy. Here, immunecells are changed (1) intrinsically (in vivo modification which wouldinclude vaccination-based approaches and the like), (2) extrinsicallyvia adoptive cell therapies or immune cell modification strategies, suchas via CAR-T therapy, immune cell grafting, immune cell transplantation,or stem cell transplantation, and related strategies, and anycombination of intrinsic or extrinsic modification.

The immune-modulating therapy may be an antagonist for immune checkpointinhibition. The immune-modulating therapy may be an agonist for immuneco-stimulation signal. The immune-modulating therapy may be astimulatory factor affecting immune cell priming and activation. Theimmune-modulating therapy may be a chemotactic agent. Theimmune-modulating therapy may be a cytokine-related immune modulator.The immune-modulating therapy may be chemotherapeutic immunestimulation. The immune-modulating therapy may be radiotherapeuticimmune stimulation. The immune-modulating therapy may be a vaccine. Theimmune-modulating therapy may be activation of an adaptive immuneresponse. The immune-modulating therapy may be activation of innateimmune response.

The immune-modulating therapy may be an antagonist for immune checkpointinhibition having a target selected from the group consisting of PD-1,PD-L1, CTLA-4, LAG3, TIM-2, CD47, KIR, TIM3, and CD30.

The target may be programmed cell death protein 1 (PD-1), also known asand CD279 (cluster of differentiation 279). PD-1 is a cell surfacereceptor that downregulates the immune system and promotesself-tolerance by suppressing T cell inflammatory activity. PD-1 is animmune checkpoint and guards against autoimmunity through a dualmechanism of promoting apoptosis (programmed cell death) inantigen-specific T-cells in lymph nodes while simultaneously reducingapoptosis in regulatory T cells (anti-inflammatory, suppressive Tcells). PD-1 inhibits the immune system. This prevents autoimmunediseases, but it can also prevent the immune system from killing cancercells. Drugs that block PD-1, the PD-1 inhibitors, activate the immunesystem to attack tumors PD-1 is a cell surface receptor that belongs tothe immunoglobulin superfamily and is expressed on T cells and pro-Bcells. PD-1 binds two ligands, PD-L1 and PD-L2.

The target may be programmed death-ligand 1 (PD-L1). Also known ascluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H), PD-L1 isa protein that in humans is encoded by the CD274 gene. Programmeddeath-ligand 1 (PD-L1) is a 40 kDa type 1 transmembrane protein that maysuppress the immune system during pregnancy, tissue allografts,autoimmune disease, and hepatitis. Normally, the immune system reacts toforeign antigens associated with exogenous or endogenous danger signals,which trigger a proliferation of antigen-specific CD8+ T cells and/orCD4+ helper cells. The binding of PD-L1 to PD-1 or B7.1 transmits aninhibitory signal that reduces the proliferation of these T cells andcan also induce apoptosis, which is further mediated by a lowerregulation of the gene Bcl-2.

The target may be cytotoxic T-lymphocyte-associated protein 4 (CTLA-4),also known as CD152 (cluster of differentiation 152). CTLA-4 is aprotein receptor that, functioning as an immune checkpoint,downregulates immune responses. CTLA4 is constitutively expressed inregulatory T cells but only upregulated in conventional T cells afteractivation.

The target may be lymphocyte-activation gene 3 (LAG-3), a protein thatin humans is encoded by the LAG3 gene. LAG3 is also designated CD223(cluster of differentiation 223). LAG3 is a cell surface molecule withdiverse biologic effects on T cell function. It is an immune checkpointreceptor.

The target may be T cell immunoglobulin and mucin domain family 2(TIM-2) for family 3 (TIM3).

The target may be a Cluster of Differentiation 47 (CD47), also known asintegrin associated protein (IAP). CD47 is a transmembrane protein that,in humans, is encoded by the CD47 gene. CD47 belongs to theimmunoglobulin superfamily and partners with membrane integrins andbinds the ligands thrombospondin-1 (TSP-1) and signal-regulatory proteinalpha (SIRPα). This is because the protein IAP produced by CD-47 acts asa don't-eat-me signal to the immune system and drives organ fibrosis.CD47 is involved in many cellular processes, including apoptosis,proliferation, adhesion, and migration. Furthermore, it plays a key rolein immune and angiogenic responses. CD47 is ubiquitously expressed inhuman cells and has been found to be overexpressed in many differenttumor cells. Expression in equine cutaneous tumors has been reported aswell.

The target may be killer-cell immunoglobulin-like receptors (KIR), afamily of type I transmembrane glycoproteins expressed on the plasmamembrane of natural killer (NK) cells, and a minority of T cells.

The target may be CD30. CD30, also known as TNFRSF8, is a cell membraneprotein of the tumor necrosis factor receptor family and tumor marker.This receptor is expressed by activated, but not by resting, T, and Bcells. TRAF2 and TRAF5 can interact with this receptor and mediate thesignal transduction that leads to the activation of NF-kappaB. It is apositive regulator of apoptosis, limits the proliferative potential ofautoreactive CD8 effector T cells, and protects the body againstautoimmunity. Two alternatively spliced transcript variants of this geneencoding distinct isoforms have been reported.

The immune-modulating therapy may be an agonist for immuneco-stimulation signal having a target selected from the group consistingof CD137/41BB, 41BBL, OX40, CD27, CD40/CD40L/cd40/CEA-CD3CD, and STING.

The target may be CD137/41BB. CD137 is a member of the tumor necrosisfactor (TNF) receptor family. Its alternative names are tumor necrosisfactor receptor superfamily member 9 (TNFRSF9), 4-1BB, and induced bylymphocyte activation (ILA). CD137 can be expressed by activated Tcells, but on CD8 than on CD4 T cells. In addition, CD137 expression isfound on dendritic cells, B cells, follicular dendritic cells, naturalkiller cells, granulocytes, and cells of blood vessel walls at sites ofinflammation.

The target may be 4-1BB, a type 2 transmembrane glycoprotein belongingto the TNF superfamily, expressed on activated T lymphocytes. 41BBL isthe ligand for 4-1BB.

The target may be OX40. Tumor necrosis factor receptor superfamily,member 4 (TNFRSF4), also known as CD134 and OX40 receptor, is a memberof the TNFR-superfamily of receptors, which is not constitutivelyexpressed on resting naïve T cells, unlike CD28. OX40 is a secondaryco-stimulatory immune checkpoint molecule, expressed after 24 to 72hours following activation; its ligand, OX40L, is also not expressed onresting antigen-presenting cells but is following their activation.Expression of OX40 is dependent on full activation of the T cell;without CD28, expression of OX40 is delayed and of fourfold lowerlevels.

The target may be a cluster of differentiation 27 (CD27), a member ofthe tumor necrosis factor receptor superfamily, and a co-stimulatoryimmune checkpoint molecule. The protein encoded by this gene is a memberof the TNF-receptor superfamily. This receptor is needed for thegeneration and long-term maintenance of T cell immunity. It binds toligand CD70 and regulates B-cell activation and immunoglobulinsynthesis. This receptor transduces signals that lead to the activationof NF-κB and MAPK8/JNK. Adaptor proteins TRAF2 and TRAF5 have been shownto mediate the signaling process of this receptor. CD27-binding protein(SIVA), a proapoptotic protein, can bind to this receptor and inducesapoptosis by this receptor.

The target may be a cluster of differentiation 40 or its ligandCD40/CD40L/cd40/CEA-CD3CD. CD40 is a costimulatory protein found onantigen-presenting cells and is needed for their activation. BindingCD154 (CD40L) on TH cells to CD40 activates antigen-presenting cells andinduces a variety of downstream effects. Deficiency can cause Hyper-IgMsyndrome type 3.

The target may be a stimulator of interferon genes (STING), also knownas transmembrane protein 173 (TMEM173) and MPYS/MITA/ERIS. STING is aprotein that in humans is encoded by the TMEM173 gene.

The immune-modulating therapy may be a stimulatory factor affectingimmune cell priming and activation selected from the group consisting ofCD28/7.1, CD137/CD137L, OX40/OX40L, CD27/CD70, HVEM, GITR, CDN, ATB,HMGB1, TLR4, LR7, TLR 8, TLR9, MICA/MICB, B7-H2, B7-H3, B7-H4, andB7-1/2.

The stimulatory factor may be a Cluster of Differentiation28(CD28/B7.1), one of the proteins expressed on T cells that provideco-stimulatory signals for T cell activation and survival. T cellstimulation through CD28, in addition to the T-cell receptor (TCR), canprovide a potent signal for producing various interleukins (IL-6 inparticular). CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2)proteins. When activated by Toll-like receptor (TLR) ligands, the CD80expression is upregulated in antigen-presenting cells (APCs). The CD86expression on antigen-presenting cells is constitutive (the expressionis independent of environmental factors). CD28 is the only B7 receptorconstitutively expressed on naive T cells. Association of the TCR of anaive T cell with MHC:antigen complex without CD28:B7 interactionresults in a T cell that is anergic.

The stimulatory factor may be CD137/CD137L. CD137 is a member of thetumor necrosis factor (TNF) receptor family. Its alternative names aretumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-1BB,and induced by lymphocyte activation (ILA). CD137 is a co-stimulatoryimmune checkpoint molecule.

The stimulatory factor may be OX40/OX40L. The stimulatory factor may beCD27/CD70.

The stimulatory factor may be herpesvirus entry mediator (HVEM), alsoknown as tumor necrosis factor receptor superfamily member 14(TNFRSF14), which is a human cell surface receptor of the TNF-receptorsuperfamily.

The stimulatory factor may be GITR. Tumor necrosis factor receptorsuperfamily member 18 (TNFRSF18), also known as activation-inducibleTNFR family receptor (AITR) or glucocorticoid-induced TNFR-relatedprotein (GITR), is a protein that in humans is encoded by the TNFRSF18gene. GITR is a co-stimulatory immune checkpoint molecule.

The stimulatory factor may be CDN. The stimulatory factor may be ATB.

The stimulatory factor may be a high mobility group box 1 protein, alsoknown as high-mobility group protein 1 (HMG-1), and amphoterin, aprotein that in humans is encoded by the HMGB1 gene. In the nucleus,HMGB1 interacts with nucleosomes, transcription factors, and histones.This nuclear protein organizes the DNA and regulates transcription.After binding, HMGB1 bends DNA, which aids the binding of otherproteins. HMGB1 supports the transcription of many genes in interactionswith many transcription factors. It also interacts with nucleosomes toloosen packed DNA and remodel the chromatin. Contact with core histoneschanges the structure of nucleosomes. HMGB1 in the nucleus depends onposttranslational modifications. When the protein is not acetylated, itstays in the nucleus, but hyperacetylation on lysine residues causes itto translocate into the cytosol. HMGB1 has been shown to play animportant role in helping the RAG endonuclease form a paired complexduring V(D)J recombination. HMG-1 belongs to the high mobility group andcontains the HMG-box domain.

The stimulatory factor may be toll-like receptor 4 (TLR4). Atransmembrane member of the toll-like receptor family, which belongs tothe pattern recognition receptor (PRR) family. Its activation leads toan intracellular signaling pathway NF-κB and inflammatory cytokineproduction responsible for activating the innate immune system. TLR4 ismost well-known for recognizing lipopolysaccharide (LPS), a componentpresent in many Gram-negative bacteria (e.g., Neisseria spp.) and selectGram-positive bacteria. Its ligands also include several viral proteins,polysaccharide, and a variety of endogenous proteins such as low-densitylipoprotein, beta-defensins, and heat shock protein. TLR4 has also beendesignated as CD284 (cluster of differentiation 284).

The stimulatory factor may be toll-like receptor 7 (TLR7), a proteinthat in humans is encoded by the TLR7 gene. TLR7 recognizessingle-stranded RNA in endosomes, a common feature of viral genomes thatare internalized by macrophages and dendritic cells. TLR7 recognizessingle-stranded RNA of viruses such as HIV and HCV. TLR7 can recognizeGU-rich single-stranded RNA. However, the presence of GU-rich sequencesin the single-stranded RNA is not enough to stimulate TLR7.

The stimulatory factor may be toll-like receptor 8 (TLR8). Also known ascluster of differentiation 288 (CD288), TLR8 a protein that in humans isencoded by the TLR8 gene. TLR8 can recognize GU-rich single-strandedRNA. However, the presence of GU-rich sequences in the single-strandedRNA is not enough to stimulate TLR8. TLR8 recognizes G-richoligonucleotides. TLR8 is an endosomal receptor that recognizessingle-stranded RNA (ssRNA) and can recognize ssRNA viruses such asInfluenza, Sendai, and Coxsackie B viruses. TLR8 binding to the viralRNA recruits MyD88 and leads to activation of the transcription factorNF-κB and antiviral response. TLR8 recognizes single-stranded RNA ofviruses such as HIV and HCV.

The stimulatory factor may be toll-like receptor 9 (TLR9), also known asCD289 (cluster of differentiation 289). TLR9 is a member of thetoll-like receptor (TLR) family. TLR9 is an important receptor expressedin immune system cells, including dendritic cells, macrophages, naturalkiller cells, and other antigen-presenting cells. TLR9 preferentiallybinds DNA present in bacteria and viruses and triggers signalingcascades that lead to a pro-inflammatory cytokine response. Cancer,infection, and tissue damage can all modulate TLR9 expression andactivation. TLR9 is also an important factor in autoimmune diseases, andthere is active research into synthetic TLR9 agonists and antagoniststhat help regulate autoimmune inflammation.

The stimulatory factor may be major histocompatibility complex (MHC)class I polypeptide-related sequence A (MICA) is a cell surfaceglycoprotein encoded by the MICA gene within the MHC locus. MICA isrelated to MHC class I and has a similar domain structure, which is madeup of an external α1α2α3 domain, a transmembrane segment, and aC-terminal cytoplasmic tail. However, MICA is not associated with02-microglobulin nor binds peptides as conventional MHC class Imolecules do. MICA works as a stress-induced ligand for the NKG2Dreceptor. For example, the heat shock stress pathway regulates MICAexpression as transcription of MICA is regulated by promoter heat shockelement. MICA is broadly recognized by NK cells, γδ T cells, and CD8+ αβT cells, which carry NKG2D receptor on their cell surface. Because ofNKG2D-MICA engagement, effector cytolytic responses of T cells and NKcells against epithelial tumor cells expressing MICA are started.

The stimulatory factor may be major histocompatibility complex (MHC)class I polypeptide-related sequence B (MICB) is a protein that isencoded by the MICB gene within the MHC locus. MICB is related to MHCclass I and has a similar domain structure, which is made up of anexternal α1α2α3 domain, a transmembrane segment, and a C-terminalcytoplasmic tail. MICB is a stress-induced ligand for the NKG2Dreceptor. The heat shock stress pathway is involved in the regulation ofMICB expression as transcription of MICB is regulated by the promoterheat shock element.

The stimulatory factor may be a B7 protein. B7 is a type of peripheralmembrane protein found on activated antigen-presenting cells (APC) that,when paired with either a CD28 or CD152 (CTLA-4) surface protein on a Tcell, can produce a costimulatory signal or a coinhibitory signal toenhance or decrease the activity of an MHC-TCR signal between the APCand the T cell, respectively. Binding the B7 of APC to CTLA-4 of T-cellscauses inhibition of the activity of T-cells. There are two major typesof B7 proteins: B7-1 or CD80 and B7-2 or CD86. However, it is not knownif they differ significantly from each other. CD28 and CTLA-4 eachinteract with both B7-1 and B7-2. The stimulatory factor may be B7-H2.The stimulatory factor may be B7-H3. The stimulatory factor may beB7-H4. The stimulatory factor may be B7-1/2.

The immune-modulating therapy may be a chemotactic agent selected fromthe group consisting of CX3CL1, CXCL9, CXCL10, CCL5, LFA1, ICAM1,selectin E, selectin P, selectin N, CXCR4, CCR2, CCL21, CCR5, CXCR1,CXCR2, CSF1R, and CCR4. The chemotactic agent may be a chemokine, asmall protein that regulates cell trafficking of leukocytes. Thechemokines also play fundamental roles in the development, homeostasis,and function of the immune system, and they have effects on cells of thecentral nervous system as well as on endothelial cells involved inangiogenesis or angiostasis.

The chemotactic agent may be CX3CL1. Fractalkine, also known aschemokine (C-X3-C motif) ligand 1, is a protein that in humans isencoded by the CX3CL1 gene. Fractalkine is a large cytokine protein of373 amino acids. It has multiple domains and is the only known member ofthe CX3C chemokine family. The polypeptide structure of CX3CL1 differsfrom the typical structure of other chemokines.

The chemotactic agent may be chemokine (C-X-C motif) ligand 9 (CXCL9), asmall cytokine belonging to the CXC chemokine family that is also knownas monokine induced by gamma interferon (MIG). CXCL9 is a T-cellchemoattractant, which is induced by IFN-γ. It is closely related to twoother CXC chemokines called CXCL10 and CXCL11, whose genes are near thegene for CXCL9 on human chromosome 4. CXCL9, CXCL10, and CXCL11 allelicit their chemotactic functions by interacting with the chemokinereceptor CXCR3. Neutrophil collagenase/matrix metalloproteinase 8(MMP-8) degrades CXCL9 and cleaves CXCL10 at two positions. GelatinaseB/matrix metalloproteinase 9 (MMP-9) degrades CXCL10 and cleaves CXCL9at three different sites in its extended carboxy-terminal region.

The chemotactic agent may be C-X-C motif chemokine 10 (CXCL10), alsoknown as interferon gamma-induced protein 10 (IP-10) or small-induciblecytokine B10. CXCL10 is an 8.7 kDa protein that, in humans, is encodedby the CXCL10 gene.

The chemotactic agent may be chemokine (C-C motif) ligand 5 (CCL5), aprotein that in humans is encoded by the CCL5 gene. It is also known asRANTES (regulated on activation, normal T cell expressed and secreted).

The chemotactic agent may be lymphocyte function-associated antigen 1(LFA-1), found on T-cells, B-cells, macrophages, neutrophils, and NKcells. LFA1 is involved in recruitment to the site of infection. Itbinds to ICAM-1 on antigen-presenting cells and functions as an adhesionmolecule. LFA-1 is the first to bind T-cells to antigen-presenting cellsand initially binds weakly. A signal from the T-cell receptor and/or thecytokine receptor changes the conformation and prolongs the cellcontact, allowing the T-cell to proliferate. LFA-1/ICAM-1 interactionleads to further T cell differentiation. LFA-1 is part of the family ofleukocyte integrins recognized by their common β-chains (α2, CD18).LFA-1 also has a distinct α-chain (αL, CD11a).

The chemotactic agent may be Intercellular Adhesion Molecule 1 (ICAM-1),also known as CD54 (Cluster of Differentiation 54). ICAM-1 is a proteinthat in humans is encoded by the ICAM1 gene, giving a cell surfaceglycoprotein typically expressed on endothelial cells and cells of theimmune system. It binds to integrins of type CD11a/CD18 or CD11b/CD18and is also exploited by rhinovirus as a receptor.

The chemotactic agent may be one or more selectins. The selectins(cluster of differentiation 62 or CD62) are a family of cell adhesionmolecules (or CAMs). All selectins are single-chain transmembraneglycoproteins that share similar properties to C-type lectins due to arelated amino terminus and calcium-dependent binding. Selectins bind tosugar moieties, and so are a type of lectin, cell adhesion proteins thatbind sugar polymers. The chemotactic agent may be selectin E. Thechemotactic agent may be selectin P. The chemotactic agent may beselectin N.

The chemotactic agent may be CXCR4. C-X-C chemokine receptor type 4(CXCR-4), also known as fusin or CD184 (cluster of differentiation 184),is a protein that in humans is encoded by the CXCR4 gene.

The chemotactic agent may be C-C chemokine receptor type 2 (CCR2), alsoknown as cluster of differentiation 192 (CD192), a protein that inhumans is encoded by the CCR2 gene. CCR2 is a chemokine receptor. Thisgene encodes two isoforms of a receptor for monocyte chemoattractantprotein-1 (CCL2), a chemokine that specifically mediates monocytechemotaxis. Monocyte chemoattractant protein-1 is involved in monocyteinfiltration in inflammatory diseases, such as rheumatoid arthritis andin the inflammatory response against tumors. The receptors encoded bythis gene mediate agonist-dependent calcium mobilization and inhibitionof adenylyl cyclase.

The chemotactic agent may be chemokine (C-C motif) ligand 21 (CCL21), asmall cytokine belonging to the CC chemokine family. This chemokine isalso known as 6Ckine (because it has six conserved cysteine residuesinstead of the four cysteines typical to chemokines), exodus-2, andsecondary lymphoid tissue chemokine (SLC). The gene for CCL21 is onhuman chromosome 9. CCL21 elicits its effects by binding to a cellsurface chemokine receptor known as CCR7.

The chemotactic agent may be C-C chemokine receptor type 5 (CCR5), alsoknown as cluster of differentiation 195 (CD195), a protein on thesurface of white blood cells involved in the immune system, as it actsas a receptor for chemokines. This is the process by which T cells areattracted to specific tissue and organ targets. Many forms of HIV, thevirus that causes AIDS, initially use CCR5 to enter and infect hostcells. Certain individuals carry a CCR5-Δ32 mutation in the CCR5 gene,protecting them against these strains of HIV.

The chemotactic agent may be C-X-C motif chemokine receptor 1 (CXCR1),also known as interleukin 8 receptor, alpha (IL8RA), and a cluster ofdifferentiation 181 (CD181). The protein encoded by this gene is amember of the G-protein-coupled receptor family. This protein is areceptor for interleukin 8 (IL8). It binds to IL8 with high affinity andtransduces the signal through a G-protein-activated second messengersystem. Knockout studies in mice suggested that this protein inhibitsembryonic oligodendrocyte precursor migration in the developing spinalcord. This gene, IL8RB, a gene encoding another high-affinity IL8receptor, and IL8RBP, a pseudogene of IL8RB, form a gene cluster in aregion mapped to chromosome 2q33-q36. Stimulation of CXCR1 inneutrophils by its primary ligand, Interleukin 8, leads to neutrophilchemotaxis and activation.

The chemotactic agent may be CXCR2, also known as interleukin 8receptor, beta (IL8RB). The protein encoded by this gene is a member ofthe G-protein-coupled receptor family. This protein is a receptor forinterleukin 8 (IL8). It binds to IL8 with high affinity and transducesthe signal through a G-protein-activated second messenger system(Gi/o-coupled). This receptor also binds to chemokine (C-X-C motif)ligand 1 (CXCL1/MGSA), a protein with melanoma growth stimulatingactivity, and is a major component for serum-dependent melanoma cellgrowth. In addition, it binds ligands CXCL2, CXCL3, and CXCL5. Theangiogenic effects of IL8 in intestinal microvascular endothelial cellsare found to be mediated by this receptor. Knockout studies in micesuggested that this receptor controls the positioning of oligodendrocyteprecursors in developing spinal cord by arresting their migration. Thisgene, IL8RA, a gene encoding another high-affinity IL8 receptor, andIL8RBP, a pseudogene of IL8RB, form a gene cluster in a region mapped tochromosome 2q33-q36. Mutations in CXCR2 cause hematological traits.

The chemotactic agent may be a colony-stimulating factor 1 receptor(CSF1R), also known as macrophage colony-stimulating factor receptor(M-CSFR), and CD115 (Cluster of Differentiation 115), a cell-surfaceprotein encoded, in humans, by the CSF1R gene (also known as c-FMS). Itis a receptor for a cytokine called colony-stimulating factor 1. Theencoded protein is a single-pass type I membrane protein and acts as thereceptor for colony-stimulating factor 1, a cytokine that controls theproduction, differentiation, and function of macrophages. This receptormediates most, if not all, of the biological effects of this cytokine.Ligand binding activates CSF1R through a process of oligomerization andtrans-phosphorylation. The encoded protein is a tyrosine kinasetransmembrane receptor and member of the CSF1/PDGF receptor family oftyrosine-protein kinases.

The chemotactic agent may be CCR4. C-C chemokine receptor type 4 is aprotein that in humans is encoded by the CCR4 gene. CCR4 has alsorecently been designated CD194 (cluster of differentiation 194). Theprotein encoded by this gene belongs to the G protein-coupled receptorfamily. It is a receptor for the CC chemokines CCL2 (MCP-1), CCL4(MIP-1), CCL5 (RANTES), CCL17 (TARC), and CCL22 (Macrophage-derivedchemokine).

The immune-modulating therapy may be a chemotactic agent comprising acytokine selected from the group consisting of IL-1, IL-2, IL-4, IL-6,IL-7, IL-8, IL-13, IL-12, interferon gamma, IFNa, TNFa, CSF1, CSF1R, andGM-CSF.

The chemotactic agent may be the interleukin-1 family (IL-1 family), agroup of 11 cytokines, which regulate immune and inflammatory responsesto infections or sterile insults.

The chemotactic agent may be interleukin 2 (IL-2), a cytokineglycoprotein that stimulates the growth of T cell lymphocytes andprovides other biochemical signaling to the immune system.

The chemotactic agent may be interleukin 4 (IL4, IL-4), a cytokine thatinduces differentiation of naive helper T cells (Th0 cells) to Th2cells. Upon activation by IL-4, Th2 cells then produce more IL-4 in apositive feedback loop. Basophils may initially produce IL-4, thusinducing Th0 differentiation. IL-2 is closely related and has functionslike interleukin 13.

The chemotactic agent may be interleukin 6 (IL-6), a pro-inflammatorycytokine.

The chemotactic agent may be interleukin 7 (IL-7), a protein that inhumans is encoded by the IL7 gene. IL-7 is a hematopoietic growth factorsecreted by stromal cells in the bone marrow and thymus. IL-7 is alsoproduced by keratinocytes, dendritic cells, hepatocytes, neurons, andepithelial cells, but not by normal lymphocytes.

The chemotactic agent may be interleukin 8 (IL-8), a chemokine of theimmune system

The chemotactic agent may be interleukin 13 (IL-13), a protein that inhumans is encoded by the IL13 gene. IL-13 is on chromosome 5q31, with alength of 1.4 kb. IL-13 and IL-4 show a 30% sequence similarity and havea similar structure. IL-13 is a cytokine secreted by many cell types,but especially T helper type 2 (Th2) cells; that is, a mediator ofallergic inflammation and disease.

The chemotactic agent may be interleukin 12 (IL-12), an interleukinnaturally produced by dendritic cells, macrophages, neutrophils, andhuman B-lymphoblastoid cells (NC-37) in response to antigenicstimulation. IL-12 is involved in the differentiation of naive T cellsinto Th1 cells. IL-12 is known as a T cell-stimulating factor, which canstimulate the growth and function of T cells. It stimulates theproduction of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha(TNF-α) from T cells and natural killer (NK) cells and reduces IL-4mediated suppression of IFN-γ. T cells that produce IL-12 have acoreceptor, CD30, which is associated with IL-12 activity.

The chemotactic agent may be interferon gamma (IFNγ), a dimerizedsoluble cytokine, and is the only member of the type II class ofinterferons.

The chemotactic agent may be IFN-α, which are proteins that are producedby leukocytes. They are involved in an innate immune response againstviral infection. The genes responsible for their synthesis come in 13subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7,IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes arefound together in a cluster on chromosome 9. The recombinant type isinterferon alfacon-1. The pegylated types are pegylated interferonalfa-2a and pegylated interferon alfa-2b.

The chemotactic agent may be tumor necrosis factor (TNF, tumor necrosisfactor-alpha, TNFα, cachexin, or cachectin), a cell-signaling protein(cytokine) involved in systemic inflammation. TNFa is one of thecytokines that make up the acute phase reaction. TNFα is producedchiefly by activated macrophages, although it can be produced by manyother cell types such as CD4+ lymphocytes, NK cells, neutrophils, mastcells, eosinophils, and neurons. TNF primarily regulates immune cells.TNF, being an endogenous pyrogen, can induce fever, apoptotic celldeath, cachexia, inflammation, and to inhibit tumorigenesis and viralreplication, and respond to sepsis via IL1 and IL6 producing cells.Dysregulation of TNF production has been implicated in a variety ofhuman diseases, including Alzheimer's disease, cancer, major depression,psoriasis, and inflammatory bowel disease (IBD). Though controversial,studies of depression and IBD are linked to TNF levels. Recombinant TNFis used as an immunostimulant under the INN tasonermin. TNF can beproduced ectopically in the setting of malignancy and parallelsparathyroid hormone both in causing secondary hypercalcemia and in thecancers with which excessive production is associated.

The chemotactic agent may be CSF1. The chemotactic agent may be CSF1R.

The chemotactic agent may be a granulocyte-macrophage colony-stimulatingfactor (GM-CSF), also known as colony-stimulating factor 2 (CSF2), amonomeric glycoprotein secreted by macrophages, T cells, mast cells,natural killer cells, endothelial cells, and fibroblasts that functionsas a cytokine. The pharmaceutical analogs of naturally occurring GM-CSFare called sargramostim and molgramostim. Unlike the granulocytecolony-stimulating factor, which specifically promotes neutrophilproliferation and maturation, GM-CSF affects more cell types, especiallymacrophages and eosinophils.

The immune-modulating therapy may be chemotherapeutic immune stimulationselected from the group consisting of cyclophosphamide, paclitaxel,doxorubicin, TDO2, IDO, ARG1, ARG2, PDE5, P2X7 inhibitor, P2Y11inhibitor, A2A Receptor inhibitor, A2B Receptor inhibitor, COX2inhibitor, EP2 receptor antagonist, EP4 receptor antagonist, RON kinaseinhibitor, an ALK5 kinase inhibitor, CSF1 kinase inhibitor, PI3K deltakinase inhibitor, PI3K gamma kinase inhibitor, BRAF V600E kinaseinhibitor, arginase, and iNOS.

The immune-modulating therapy may be radiotherapeutic immune stimulationselected from the group consisting of gamma irradiation, external beamradiotherapy, stereotactic radiotherapy, radiosurgery, virtualsimulation, 3-dimensional conformal radiation therapy,intensity-modulated radiation therapy, intensity-modulated radiationtherapy (IMRT), volumetric modulated arc therapy (VMAT), particletherapy, proton beam therapy, auger therapy, brachytherapy,intraoperative radiotherapy, radioisotope therapy, and beta irritation.

The immune-modulating therapy may be a vaccine to TLR4 or TLR9. Thevaccine may be toll-like receptor 4 (TLR4). The vaccine may be toll-likereceptor 9 (TLR9).

The method may further comprise assessing whether a lymphatic system ina subject is dysregulated. Any method described herein may be used toassess the functioning of the lymphatic system.

D. Cancer

The cancer may be selected from the group consisting of lung cancer,breast cancer, a cancer of the gastrointestinal tract, a cancer ofunknown origin, head and neck cancer, bladder cancer, prostate cancer,skin cancer, kidney cancer, a primary brain tumor, ocular tumor,sarcoma, a cancer of primary soft tissue, mesenchymal cancer, bonecancer, a tumor of the lymphatic system, and leukemia.

The cancer may be lung cancer selected from the group consisting ofnon-small cell lung cancer (NSCLC), squamous cell lung cancer, largecell lung cancer, small cell lung cancer, bronchogenic carcinoma,adenocarcinoma, neuroendocrine lung cancer, and bronchoalveolar lungcancer.

The cancer may be breast cancer selected from the group consisting ofductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC),lobular carcinoma (ILC), inflammatory breast cancer, lobular carcinomain situ (LCIS), male breast cancer, Paget's disease of the nipple, andPhyllodes tumors of the breast. The breast cancer may be invasive ductalcarcinoma selected from the group consisting of tubular carcinoma of thebreast, medullary carcinoma of the breast, mucinous carcinoma of thebreast, papillary carcinoma of the breast, and cribriform carcinoma ofthe breast. The cancer may be breast cancer defined by hormone receptorstatus selected from the group consisting of estrogen receptor-positive,estrogen receptor-negative, progesterone receptor-positive, progesteronereceptor-negative, Herceptin positive, Herceptin negative, andcombinations thereof. Cancer may be breast cancer defined by expressionof a pre-defined set of genes selected from the group consisting ofmammaprint, oncotypeDX, intrinsic subtypes, and nanostring prosigna.

The cancer may be a cancer of the gastrointestinal tract selected fromthe group consisting of a tumor of the stomach, gastric cancer, duodenalcancer, small or large intestine cancer, colorectal cancer, anal cancer,liver cancer, pancreatic cancer, gall bladder cancer,cholangiocarcinoma, and neuroendocrine cancer.

The cancer may be a skin cancer selected from the group consisting ofbasal cell cancer, squamous cell cancer, and melanoma.

The cancer may be kidney cancer selected from the group consisting ofrenal cell cancer and oncocytoma.

The cancer may be a primary brain tumor, selected from the groupconsisting of glioma, a tumor with gliomatous components, a tumor withneuronal components, a tumor with oligodendroglial components,oligodendroglioma, astrocytoma, and glioblastoma multiforme.

The cancer may be a tumor of the lymphatic system selected from thegroup consisting of B cell lymphoma, T cell lymphoma, diffuse B celllymphoma, and Hodgkin's lymphoma.

The cancer may be leukemia selected from the group consisting of acutelymphoblastic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia, and chronic myelogenous leukemia.

The cancer may be lung cancer, and the method further may compriseadministering one or more drugs selected from the group consisting ofafatinib dimaleate, alectinib, bevacizumab, carboplatin, ceritinib,crizotinib, docetaxel, doxorubicin, erlotinib, etoposide, everolimus,gefitinib, gemcitabine, mechlorethamine, methotrexate, necitumumab,nivolumab, osimertinib, paclitaxel, paclitaxel albumin-stabilizednanoparticles, pembrolizumab, pemetrexed, ramucirumab, topotecan,vinorelbine, pharmaceutically acceptable salts thereof, and combinationsthereof.

The cancer may be advanced cancer or metastatic cancer.

F. Chemotherapeutic Agents

The methods described herein may be conducted in combination withadministering one or more chemotherapeutic agents. Non-limiting examplesof chemotherapeutic compounds which can be used in combinationtreatments include, for example, aminoglutethimide, amsacrine,anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin,busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramnustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

These chemotherapeutic compounds may be categorized by their mechanismof action into, for example, following groups:anti-metabolites/anti-cancer agents, such as pyrimidine analogs(5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine)and purine analogs, folate antagonists and related inhibitors(mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine(cladribine)); antiproliferative/antimitotic agents including naturalproducts such as vinca alkaloids (vinblastine, vincristine, andvinorelbine), microtubule disruptors such as taxane (paclitaxel,docetaxel), vincristine, vinblastine, nocodazole, epothilones andnavelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damagingagents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan,campothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide,cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin,hexamethyhnelamineoxaliplatin, iphosphamide, melphalan,merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramideand etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,streptozocin), trazenes-dacarbazinine (DTIC);antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,nilutamide) and aromatase inhibitors (letrozole, anastrozole);anticoagulants (heparin, synthetic heparin salts and other inhibitors ofthrombin); fibrinolytic agents (such as tissue plasminogen activator,streptokinase and urokinase), aspirin, dipyridamole, ticlopidine,clopidogrel, abcizimab; antimigratory agents; antisecretory agents(breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506),sirolimus (rapamycin), azathioprine, mycophenolate mofetil);antiangiogenic compounds (e.g., TNP-470, genistein, bevacizumab) andgrowth factor inhibitors (e.g., fibroblast growth factor (FGF)inhibitors); angiotensin receptor blocker; nitric oxide donors;anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleinhibitors and differentiation inducers (tretinoin); mTOR inhibitors,topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine,campothecin, daunorubicin, dactinomycin, eniposide, epirubicin,etoposide, idarubicin and mitoxantrone, topotecan, irinotecan),corticosteroids (cortisone, dexamethasone, hydrocortisone,methylprednisolone, prednisone, and prednisolone); growth factor signaltransduction kinase inhibitors; mitochondrial dysfunction inducers andcaspase activators; and chromatin disruptors.

The cancer may be lung cancer, such as non-small cell lung cancer(NSCLC). Suitable drugs for treated non-small cell lung cancer include,but are not limited to, Abitrexate™ (methotrexate), Abraxane™(paclitaxel albumin-stabilized nanoparticles), Afinitor™ (everolimus),Alecensa™ (alectinib), Alimta™ (pemetrexed disodium), Avastin™(bevacizumab), Cyramza™ (ramucirumab), Folex™ (methotrexate), Gilotrif™(afatinib dimaleate), Gemzar™ (gemcitabine hydrochloride), Iressa™(gefitinib), Keytruda™ (pembrolizumab), Mexate™ (methotrexate),Mustargen™ (mechlorethamine hydrochloride), Navelbine™ (vinorelbinetartrate), Opdivo™ (nivolumab), Paraplat™ (carboplatin), Paraplatin™(carboplatin), Portrazza™ (necitumumab), Tagrisso™, (osimertinib),Tarceva™ (erlotinib hydrochloride), Taxol™ (paclitaxel), Taxotere™(docetaxel), Xalkori™ (crizotinib), and Zykadia™ (ceritinib). Suitabledrug combinations for treating non-small ell lung cancer include, butare not limited to, carboplatin and taxol, and gemcitabine, andcisplatin.

Suitable drugs for treating small cell lung cancer include, but are notlimited to, Abitrexate™ (methotrexate), Afinitor™ (everolimus),doxorubicin hydrochloride, Etopophos™ (etoposide phosphate), etoposide,Folex™ (methotrexate), Hycamtin™ (topotecan hydrochloride), Mexate™(methotrexate), and Mustargen™ mechlorethamine hydrochloride).

Pharmaceutical compounds that can be used in combination with a VEGFR-3and a cancer immune-modulating therapy such as an immune checkpointinhibitor and (0) VEGFR-2 antagonist: (1) inhibitors of release of“angiogenic molecules,” such as bfGF (basic fibroblast growth factor);(2) neutralizers of angiogenic molecules, such as an anti-ObHGFantibody; and (3) inhibitors of endothelial cell response to angiogenicstimuli, including collagenase inhibitor, basement membrane turnoverinhibitors, angiostatic steroids, fungal-derived angiogenesisinhibitors, platelet factor 4, thrombospondin, arthritis drugs such asD-penicillamine and gold thiomalate, vitamin D3 analogs, alphainterferon, and the like.

G. Angiogenesis Inhibitors

The methods described herein may be conducted in combination withadministering one or more angiogenesis inhibitors. Compounds thatinhibit angiogenesis include, for example, endostatin protein orderivatives, lysine binding fragments of angiostatin, melanin ormelanin-promoting compounds, plasminogen fragments (e.g., Kringles 1-3of plasminogen), troponin subunits, antagonists of vitronectin, peptidesderived from Saposin B, antibiotics or analogs (e.g., tetracycline, orneomycin), dienogest-containing compositions, compounds comprising aMetAP-2 inhibitory core coupled to a peptide, the compound EM-138,chalcone, and its analogs, and naaladase inhibitors.

Depending on the combinatory therapy, administration of the polypeptidetherapeutic agents may be continued while the other therapy isadministered and/or thereafter. Administration of the therapeutic agentscan be made in a single dose or in multiple doses. In some instances,administration of the therapeutic agents can begin at least several daysprior to the conventional therapy, while in other instances, theadministration can begin either immediately before or at the time of theadministration of the conventional therapy.

Although the disclosure described herein is susceptible to variousmodifications and alternative iterations, specific embodiments thereofhave been described in greater detail above. It should be understood,however, that the detailed description of the composition is notintended to limit the disclosure to the specific embodiments disclosed.Rather, the disclosure is intended to cover all modifications,equivalents, and alternatives falling within the spirit and scope of thedisclosure as defined by the claim language.

Definitions

The compounds described herein have asymmetric centers. Compounds of thepresent disclosure having an asymmetrically substituted atom may beisolated in optically active or racemic form. All chiral,diastereomeric, racemic forms and all geometric isomeric forms of astructure are intended unless the specific stereochemistry or isomericform is specifically indicated.

“Inhibiting an established tumor metastasis” refers to decreasing thesize and/or rate of growth of metastasis, which has already beenestablished. Established metastases include metastases in lymph nodes(regional metastases) and distant organs (systemic metastases).

“Lymphangiogenesis” refers to the growth of new lymphatic vessels.

“Therapeutically effective” applied to dose or amount refers to thatquantity of a pharmaceutical composition sufficient to result in adesired therapeutic activity upon administration to a subject in needthereof, or sufficient to reduce or eliminate at least one symptom ofthe disease being treated.

“Subject” means any animal, including mammals. The term may refer to ahuman, a non-human primate, a bovine, an ovine, an equine, a porcine, acanine, a feline, or a rodent (mouse or rat).

When introducing elements of the present disclosure or theembodiments(s) thereof, the articles “a,” “an,” “the,” and “said” areintended to mean that there are one or more of the elements. The terms“comprising,” “including,” and “having” are intended to be inclusive andmean that there may be other elements other than the listed elements.

Having described the disclosure in detail, it will be apparent thatmodifications and variations are possible without departing from thescope of the disclosure defined in the appended claims.

EXAMPLES

The following examples are included to show certain embodiments of thedisclosure. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples represent techniques discoveredby the inventors to function well in the practice of the disclosure.Those of skill in the art should, however, considering the presentdisclosure, appreciate that many changes can be made in the specificembodiments disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the disclosure, therefore allmatter set forth is to be interpreted as illustrative and not in alimiting sense.

Example 1—Evaluation of Dysregulated Lymphatic System in Lung CancerPatients Treated with PD-1 Inhibitor Immunotherapy

Patients with advanced or metastatic lung cancer were treated with aninhibitor of PD-1. Their lymphatic systems are evaluated fordysfunction. In one instance, lymphatic system evaluation was performedwith serum measurements of VEGF C, VEGF D, or midkine in patients beforestarting therapy or during treatment. In other instances, thedysfunction was measured with pretreatment imaging studies and/orsimilar imaging across the treatment with immunotherapy therapy,including either computed tomography, MRI, and nuclear medicine tests,including PET imaging or ventilation-perfusion scans.

Patients with dysregulated lymphatic systems (“biomarker positive”) didsignificantly worse across the objective measures of response based onprogression-free survival, overall survival, durable response, andobjective response as found by Response Evaluation Criteria in SolidTumors (RECIST 1.1) and by immune-related response criteria (irRC).

Only patients with measurable disease at baseline were included inprotocols where objective tumor response was the primary endpoint.“Measurable disease” was the presence of at least one measurable lesion.If the measurable disease was restricted to a solitary lesion,neoplasticity was confirmed by cytology/histology. Measurable lesionscould be measured in at least one dimension with the longest diameter ofabout 20 mm using conventional techniques or about 10 mm with spiral CTscan. Non-measurable lesions included small lesions (longest diameter<20 mm with conventional techniques or <10 mm with spiral CT scan), bonelesions, leptomeningeal disease, ascites, pleural/pericardial effusion,inflammatory breast disease, lymphangitis cutis/pulmonitis, cysticlesions, and abdominal masses not confirmed or followed by imagingtechniques. All baseline evaluations were performed as closely aspossible to the beginning of treatment and not more than 4 weeks beforethe beginning of the treatment. The same method of assessment and thesame technique characterized each reported lesion at baseline and duringfollow-up.

Conventional CT and MRI were performed with cuts of 10 mm or less inslice thickness contiguously. Spiral CT was performed using a 5-mmcontiguous reconstruction algorithm. This is applied to tumors of thechest, abdomen, and pelvis. Head and neck tumors and those ofextremities followed specific protocols. Lesions on chest X-ray wereacceptable as measurable lesions when they were defined and surroundedby aerated lung.

Tumor markers alone could not assess response. If markers were initiallyabove the upper normal limit, they must normalize for a patient to be incomplete clinical response when all lesions disappeared. Cytology andhistology differentiated between partial and complete responses. Targetlesions were selected based on size (lesions with the longest diameter)and suitability for exact repeated measurements (either by imagingtechniques or clinically). A sum of the longest diameter (LD) for alltarget lesions was calculated and reported as the baseline sum LD. Thebaseline sum LD was the reference by which the objective tumor wascharacterized. Other lesions (or sites of disease) were identified asnon-target lesions and recorded at baseline. Measurements of theselesions are not needed, but the presence or absence of each was notedthroughout follow-up.

“Complete Response” (CR) was the disappearance of all target lesions orthe disappearance of all non-target lesions and normalization of tumormarker level.

“Partial Response” (PR) was at least a 30% decrease in the sum of thelongest diameter of target lesions, referring to the baseline sumlongest diameter.

“Progressive Disease” (PD) was at least a 20% increase in the sum of thelongest diameter of target lesions, referring to the smallest sumlongest diameter recorded since starting a treatment or one or more newlesions appeared. PD is also the appearance of one or more new lesionsand/or unequivocal progression of existing non-target lesions.

“Incomplete Response” or “Stable Disease” (SD) had neither sufficientshrinkage to qualify for PR nor sufficient increase to qualify for PD,referring to the smallest sum LD since the treatment started. SD had thepersistence of one or more non-target lesions and/or maintenance oftumor marker level above the normal limits.

Although the progression of only “non-target” lesions was exceptional,in such circumstances, the opinion of the treating physician prevailed,and the review panel or the study chair later confirmed the progressionstatus. The best overall response was the best response recorded fromthe start of the treatment until disease progression/recurrence,referring to PD for the smallest measurements recorded since thetreatment started. The patient's best response assignment depends onachieving both measurement and confirmation criteria, as shown in Table1:

TABLE 1 Response measurements Target lesions Non-Target lesions NewLesions Overall response CR CR No CR CR Incomplete No PR response/SD PRNon-PD No PR SD Non-PD No SD PD Any Yes or No PD Any PD Yes or No PD AnyAny Yes PD

Patients with a global deterioration of health status needingdiscontinuation of treatment without objective evidence of diseaseprogression were classified as having “symptomatic deterioration.”Objective progression was documented even after discontinuation oftreatment. In some circumstances, it was difficult to distinguish theresidual disease from normal tissue. When the evaluation of completeresponse depended on this determination, the residual lesion wasinvestigated using a fine needle aspirate or biopsy to confirm thecomplete response status. Confirmation of objective response avoidedoverestimating the response rate seen. Where confirming the response wasnot feasible, the response reports stated such.

To be assigned a status of PR or CR, changes in tumor measurements wereconfirmed by repeat assessments no less than 4 weeks after the criteriafor response were first met. Longer intervals from the study protocolwere also proper. For SD, follow-up measurements met the SD criteria atleast once after study entry at a minimum interval (in general, not lessthan 6-8 weeks) defined in the study protocol. The duration of overallresponse was measured from the time measurement criteria were met for CRor PR, whichever status was first recorded, until the first date thatrecurrence or PD was objectively documented referring to PD for thesmallest measurements recorded since the treatment started.

SD was measured from the start of the treatment until the criteria fordisease progression were met, referring to the smallest measurementsrecorded since the treatment started. The clinical relevance of theduration of SD varied for different tumor types and grades. Therefore,it was highly recommended that the protocol specified the minimal timeinterval between two measurements for determining SD. This time intervalconsidered the expected clinical benefit, such a status brought to thestudied population.

For trials where the response rate was the primary endpoint, allresponses were reviewed by an independent expert at the study'scompletion, including a simultaneous review of the patients' files andradiological images.

All patients included in the study were assessed for response totreatment, even if treatment majorly deviated from protocol or if theywere ineligible. Each patient was assigned to (1) complete response, (2)partial response, (3) stable disease, (4) progressive disease, (5) earlydeath from malignant disease, (6) early death from toxicity, (7) earlydeath because of other cause, or (9) unknown (not assessable,insufficient data). All patients who met the eligibility criteria wereincluded in the main analysis of the response rate. Patients in responsecategories 4-9 should fail to respond to treatment (diseaseprogression). Thus, an incorrect treatment schedule or drugadministration did not exclude them from the analysis of the responserate. Precise definitions for categories 4-9 were protocol specific.

Conclusions were based on all eligible patients. Analyses of a subset ofpatients excluded those with major protocol deviations, such as earlydeath for other reasons, early discontinuation of treatment, majorprotocol violations, etc. However, the conclusion for treatment efficacywas not drawn from the analyses of subsets. The reasons for excludingpatients from the analysis were reported. Moreover, the 95% confidenceintervals were provided.

FIG. 3 shows progression-free survival (%) as a function of time in daysfor cancer treatment using immune modifying therapy. Biomarker positivepatients had a median survival of 50 days. Biomarker negative patientshad a median survival of 151 days. HR for progression or death was 0.48(95% CI, 025.-0.91), at P<0.025. Table 2 shows the Cox proportionalhazard model progression-free survival for patient subsets, includingage, race, dosing, smoking status, type of lung cancer, EGFR mutation,or prior systemic therapies. Table 2 reports the hazard ratio, standarddeviation, z value, p-value related to the absolute value of z, and the95% confidence interval.

TABLE 2 Cox Proportional Hazard Mode Progression-Free Survival (PFS) Loglikelihood = −281.59881 Prob > chi2 = 0.0468 _t Haz. Ratio Std. Err. zP > |z| [95% Conf. Interval] 2.092233 .6869697 2.25 0.025 1.099323.981952 age651 1.026221 .2587925 0.10 0.918 .6260152 1.682275racewhitelasian2hispanic3 .9691919 .1316666 −0.23 0.818 .74262991.264874 dosingq321q3102q2103 .873988 .1517212 −0.78 0.438 .62192771.228206 SmokingStatuspositiveorform .6987349 .1975865 −1.27 0.205.4014319 1.216222 TypeofLungCanceradenocompo 1.438827 .4424634 1.180.237 .7874985 2.628859 EGFRmt1mutant 1.190704 .3369266 0.62 0.537.6838242 2.073306 priorsystemictherapies1 .9473828 .2945776 −0.17 0.862.5150558 1.742596

FIG. 4 shows the objective response of partial responses or completeresponses (PR/CR). The first partial responses were denoted by ellipses.The solid triangles show ongoing responses. Biomarker negative patientshad an N=18/95 (19%). Biomarker Positive patients had an N=0/95 (0%).

FIG. 5 shows the durable clinical response for the stable disease (SD)or partial response (PR) lasting at least 180 days. Lines 1-26 werebiomarker negative patients. Line 27 was a biomarker positive patient.

As seen from the data presented above, patients without a dysregulatedlymphatic system had a strikingly significant improvement in survival,thus confirming lymphatic system status for deciding treatment responsein cancer patients treated with immune-modulating therapies. Further,statistical analysis showed that a dysregulated lymphatic system in thiscohort was the single strongest factor for predicting response. Thedysregulated lymphatic system outweighed other known importantprognostic and predictive factors, including age, race, immunotherapydosing, total tumor burden, gender, smoking and mutation status, andlung cancer cell type. (See especially Table 2.)

Example 2—Treatment of Advanced and Metastatic Lung and Breast Cancerswith Modulators of Lymphatic Biology Plus Immunotherapy Results inImproved Outcomes

One to two million cells of either LLC lung cancer or 4T1 breast cancercells are subcutaneously implanted into syngeneic mice (C57 Black 6 andBALB/c). Animals are treated with vehicle alone as a control, withchemotherapy (cisplatin), or with immunotherapy alone (anti-PD-1 mAb).Animals are imaged by intravascular ultrasound (IVUS), CT/PET, or MRI,for growth of the primary tumor and for metastatic spread of the tumor.Blood samples are obtained for a multiplexed measure of serum biomarkersof lymphatic biology function (Millipore mouse 24 plex; MAGPMAG-24K).Around Study Day 10, animals are injected with 1-1.5 million more tumorcells into the tail vein. Between Study Days 13 and 16, the primarytumor is surgically resected, and animals resume treatment.

Treatment groups include (1) anti-PD-1 mAb alone; (2) anti-PD-1-mAb pluslymphatic system modulator (anti-VEGF C or anti-VEGF R3 mAb, orSAR131675); (3) cisplatin alone; (4) lymphatic system modulator(anti-VEGF C or anti-VEGF R3 mAb, or SAR131675) plus cisplatin; and (5)negative control (no treatment).

Animals are imaged weekly, and blood for serum multiplex measurements iscontinued and evaluated. Survival studies and analyses are performed,and animals are evaluated based on overall survival, progression-freesurvival, objective and durable response, and serum biomarkermeasurements. Lung tissue, lymphatics, and regional lymph nodes andtumor tissue are concurrently evaluated upon animal sacrifice forfunctional immune evaluation and analysis of the tumor and diseaseanalysis.

Results show that animals do not respond to anti-PD-1 and show no tomarginal response to cisplatin. This effect is more pronounced in thosegroups with dysregulated lymphatic systems as measured by the imagingtests and serum biomarkers tests. However, adding a lymphatic systemmodulator to either the anti-PD-1 mAb or the cisplatin-treated groupsresults in marked significant improvement. Measured outcomes includeprogression-free survival, objective and durable responses, andimprovement in biomarker profiles.

Tumor growth is augmented in non-lymphatic system modulator treatedmice, compared to mice treated with a lymphatic system modulatorcombination. Expression of inflammatory cytokines such as IFN-γ, TNF-α,IL-2, and IL-10 in draining lymph nodes is significantly reduced innon-lymphatic modulator treated mice. Moreover, decreased levels oftumor-associated antigens are detected in draining lymph nodes in thesemice, together with impaired antigen presentation. CD8+ T cells indraining lymph nodes derived from non-lymphatic system modulator treatedmice also show significantly decreased cytotoxic activity in vitro.Finally, tumor suppression activity of CD8+ T cells derived fromnon-lymphatic system modulator treated mice was reduced when adoptivelytransferred to naive wild-type mice.

In contrast, the reverse is seen in the lymphatic system modulatortreated mice with restoration or augmentation of immune response andantigen presentation and immune cell priming. IFN-γ enzyme-linkedImmunoSpot (ELISAPOT) assay, which enables direct quantification ofimmune responses, similarly show decreased activity. Similar findingsare seen with myeloid derives suppressor cells, T regulatory cells, andinhibitory macrophages (M2), which are increased in the tumor and lymphnodes and decreased in antigen-presenting cells (e.g., cross-presentingCD8+ T cells, and dendritic cells and M1 cells in the supporting anddraining lymph nodes) in the nonlymphatic modulating system treated micethat also have worse outcomes. Additionally, lymphatic vessels showincreased expression of PD-1 and blocking PDL1 with lymphatic systemmodulators results in increased T-cell stimulation by antigen-presentinglymphatic endothelial cells (LECs) in vitro overcoming peripheral,tumor-associated lymphatic endothelium associated T-cell inhibition.

These findings support that lymphatic transport is essential ingenerating optimal tumor-specific immune responses mediated by CD8+ Tcells. Lymphatic transport is impaired in subjects with dysfunctionallymphatic systems. Concurrent treatment with a lymphatic modulator(e.g., SAR131675, or anti-VEGF-C, or Anti-VEGF R3 m AB) and either animmune modulator (e.g., checkpoint inhibitor) or chemotherapy (e.g.,cisplatin) can overcome this inhibition and results in functionalimmunological improvements.

Although animals treated with lymphatic modulator improve regardless oftreatment (such as with the concurrent treatment with immunotherapy orwith cisplatin), the degree of improvement across objective measuresdirectly correlates with underlying lymphatic dysfunction. Theseresults, therefore, confirm that lymphatic modulating agents have aprofound synergistic effect with immunotherapy and/or chemotherapy insyngeneic animal models, where these drugs alone have little to noeffect by themselves. Further, this effect is mechanistically related topriming and facilitation of the immune system and/or overcoming immunebarriers associated with or aided by the tumor.

Example 3

Treatment with lymphatic system modulators with immune-modulators orchemotherapy significantly improves objective outcomes in mice withadvanced or metastatic cancer with dysregulated lymphatic systems andprevents the progression to lymphatic carcinomatosis, a form of severelymphatic dysregulation in subjects with cancer.

The experiments from Example 2 are performed using the same animalmodels (4T1 and LLC) in syngeneic mice. Another group of tumor cells istransfected to overexpress VEGF-C to stimulate severe lymphaticdysregulation. Overexpression of VEGF C by tumor cells can result in asevere lymphatic dysregulation and an extremely aggressive lymphangiticcarcinomatosis, which is a subset of the groups of dysregulatedlymphatic disorders resistant to immunotherapy (Example 1). When treatedwith a lymphatic system modulator, outcomes are profoundly improved inboth objective and immune/functional (Example 2). In this experiment, itis evaluated whether advanced tumors with inherent lymphaticdysregulation or with exaggerated lymphatic dysregulation driven by VEGFC hyperexpression can be treated and whether treatment can prevent micewith cancers from progressing to severe lymphangitic carcinomatosis.

Treatment groups include (1) anti-PD-1 mAb alone; (2) anti-PD-1-mAb pluslymphatic system modulator (anti-VEGF C or anti-VEGF R3 mAb, orSAR131675); (3) cisplatin alone; (4) lymphatic system modulator(anti-VEGF C or anti-VEGF R3 mAb, or SAR131675) plus cisplatin; and (5)negative control (no treatment).

All mice, especially mice (both 4T1 and LLC) with dysregulated lymphaticsystems as measured by imaging, and functional assays, are highly andsignificantly responsive to lymphatic system modulators plusimmune-modulator or chemotherapy, both functionally and by objectivemeasures. (These are similar objective measures as those described inExample 2.) This finding is more pronounced in the VEGF-C overexpressinganimal models with exaggerated lymphatic dysfunction and in those withlymphatic carcinomatosis. Animals with lymphangitic carcinomatosis alsoimprove in imaging and functional measurements (Example 2) when treatedwith a lymphatic system modulator plus either immunomodulatory orchemotherapy. This therapy can potentially treat and overcome thishighly aggressive form of advanced cancer.

Further, a preventative treatment with a lymphatic system modulatoralone (SAR131675, anti-VEGF-C, or anti-VEGF R3) or with an immunemodulator or chemotherapy significantly improves objective andfunctional outcomes in these animals. The treatment prevents thedevelopment of further dysregulated lymphatic dysfunction, such that nomouse progresses to lymphangitic carcinomatosis. These studies show thatlymphatic system modulators with immune modulators or chemotherapytreated aggressive tumors, some of which had lymphatic dysfunction andaggressive lymphangitic carcinomatosis, and prevents the progression anddevelopment of lymphatic carcinomatosis.

Example 4

In examining a subset of patients from Example 1, tumors responded tocheckpoint inhibition based upon lymphatic dysfunction, independent ofPD-1/L1, DNA mismatch repair status, microsatellite instability status,or hypermutant status/tumor neoantigen burden.

Patients with advanced or metastatic lung cancer were treated with aninhibitor of PD-1. Patients with a low or negative PD-L1 tumorproportion score (TPS) were evaluated as specified by the drug label,and thus not indicated for the drug and not expected to respond. Theirlymphatic systems were evaluated for dysfunction. In one instance,lymphatic system evaluation was performed with serum measurements ofVEGF C, VEGF D, or heparin-binding factor midkine in patients beforetherapy or during treatment. In other instances, the dysfunction wasmeasured with pre-treatment imaging studies and/or similar imagingacross the treatment with immunotherapy therapy, including computedtomography, MRI, and nuclear medicine tests, including PET imaging orventilation-perfusion scan.

Evaluating objective measures of response in these patients based onprogression-free survival, overall survival, and durable and objectiveresponse (RECIST 1.1) and by immune-related response criteria (irRC)confirmed that patients with dysregulated lymphatic systems (designatedas “Biomarker Positive” groups) did significantly worse across all theseobjective measures (overall survival, progression-free survival,objective response, and durable clinical response).

Keytruda™ product label teaches to the contrary. Pembrolizumab (formerlyMK-3475 and lambrolizumab, trade name Keytruda™) is a humanized antibodyused in cancer immunotherapy. It blocks a protective mechanism on cancercells and allows the immune system to destroy those cancer cells. Ittargets the programmed cell death 1 (PD-1) receptor. It is indicated forpatients with metastatic NSCLC whose tumors have high PD-L1 expressionwith a tumor proportion score (TPS) of at least 50%, as determined by anFDA-approved test, with no epidermal growth factor receptor (EGFR) oranaplastic lymphoma kinase (ALK). Keytruda™ is also indicated forpatients with metastatic NSCLC whose tumors express PD-L1 (TPS of atleast 1%), as determined by an FDA-approved test, with diseaseprogression on or after platinum-containing chemotherapy. That is, theart teaches that patients with low PD-L1 levels cannot be treated.

Opdivo™ is a programmed death receptor-1 (PD-1) blocking antibody. Alsoknown as, nivolumab is indicated for adult and pediatric (12 years andolder) patients with microsatellite instability-high (MSI-H) or mismatchrepair deficient (dMMR) metastatic colorectal cancer that has progressedfollowing treatment with fluoropyrimidine, oxaliplatin, and irinotecan.Again, the art teaches that patients with low PD-L1 levels cannot betreated.

However, contrary to teaching in the art, patients without adysregulated lymphatic system, yet who were not expected to respondbased on their PD-L1 TPS, had a striking response to the therapy with asignificant duration of overall and progression-free survival, thusconfirming that the lymphatic system critically determined response toimmune checkpoint therapy.

Further, statistical analysis showed that the presence of a dysregulatedlymphatic system in this cohort was the single strongest factor forpredicting response, outweighing other known important prognostic andpredictive factors including age, race, immunotherapy dosing, totaltumor burden, gender, smoking, and mutation status, and lung cancer celltype. Similar results were seen in patients treated with PD-L1inhibitors, with low neoantigen burdens, hypermutant status, and thetumor types shown not to respond to checkpoint inhibition therapydescribed above.

FIG. 6 shows overall survival (% alive) as a function of time in days.Biomarker positive patients survived a median of 47 to 67 days.Biomarker negative patients survived a median of 445 days.

FIG. 7 shows progression-free survival (%) as a function of time indays. Biomarker positive patients had a median survival of 47 to 68days. Biomarker negative patients had a median survival of 189 days.

Thus, dysregulated lymphatic systems were a powerful predictor ofresponse to immune-modulating therapies independent of the art, whichteaches that PD-L1/PD-1 TPS and hypermutant/neoantigen status werecritical determinants of response. Certain tumors types, such astriple-negative breast cancer, colon, pancreatic, glioblastomamultiforme (GBM), prostate cancer, inflammatory, and ER+/HER2+ breastcancers, did not respond well to these therapies.

Example 5—Specificity of Monoclonal Antibodies Against Flt4

The purified recombinant Fms-related tyrosine kinase 4 (Flt4)extracellular domain-6×His fusion product was labeled with europium,according to Mukkala et al., Anal. Biochem, 176(2):319-325 (1989), withthe following modification: a 250 times molar excess of isothiocyanateOTTA-Eu (N1 chelate, WALLAC, Finland) was added to the Flt4 solution(0.5 mg/ml in PBS), and the pH was adjusted to about 9 by adding 0.5 Msodium carbonate buffer, pH 9.8. The labeling was performed overnight at+4° C. Unbound label was removed using PD-10 (Pharmacia, Sweden) withTSA buffer (50 mM Tris-HCl, pH 7.8, containing 0.15 M NaCl) as eluent.

After purification, 1 mg/mL bovine serum albumin (BSA) was added to thelabeled Flt4, and the label was stored at +4° C. The average number ofeuropium ions incorporated per Flt4 molecule was 1.9, as determined bymeasuring the fluorescence in a ratio to that of known EuCl3 standards(Hemmila et al., Anal. Biochem., 137:335-343 (1984)).

The antibodies were screened using a Sandwich-type immunofluorometricassay, using microtitration strip wells (NUNC, polysorb) coated withrabbit anti-mouse 1 g (Z 259, DAKOPATTS). The pre-coated wells werewashed once by Platewash 1296-024 (WALLAC) with DELFIA wash solution.The DELFIA assay buffer was used as a dilution buffer for cell culturesupernatants and for the serum of the splenectomized mouse (at dilutionsbetween 1:1000 to 1:100,000) used as a positive control in thepreliminary screening assay. An overnight incubation at +4° C. (oralternatively for 2 hours at room temperature) was begun by shaking on aPlateshake shaker (1296-001, WALLAC) for 5 minutes followed by washingfour times with wash solution as described above.

The europium-labeled Flt4 was added at a dilution of 1:500 in 100 mL ofthe assay buffer. After 5 minutes on a Plateshake shaker and one-hourincubation at room temperature, the strips were washed as describedabove.

The enhancement solution (DELFIA) was added at 200 μL/well. The plateswere then shaken for 5 minutes on a Plateshake shaker, and the intensityof fluorescence was measured by ARCUS-1230 (WALLAC) for 10-15 minutes.(Lovgren et al., In: Collins W. P. (Ed.) Alternative immunoassays, JohnWiley & Sons Ltd. (1985), pp. 203-216). The DELFIA results show that allmonoclonal antibodies tested bound the Flt4 EC antigen.

Monoclonal antibodies reactive with the Flt4 (and the hybridomas whichproduce the antibodies) were selected for further screening. Theresulting monoclonal antibodies were used in double-antibodyimmunofluorescence staining of NIH3T3 cells expressing the LTR-FLT41construct and neomycin-resistant transfected NIH3T3 cells: The cellswere detached from the culture plates using EDTA, stained, and analyzedin a fluorescence-activated cell sorter (FACS). The results of FACSanalysis are given as percentages of cells staining positive with theindicated monoclonal antibody.

The FACS results with LTR-FLT41-transfected cells indicate that theantibodies effectively recognize Flt4-expressing cells. These sameantibodies give only background staining of neomycinphosphotransferase-transfected NIH3T3 cells. Thus, the antibodiesspecifically recognize the Flt4 tyrosine kinase on the cell surface.

One clone, designated anti-Flt4 hybridoma 9D9F9, was found to stablysecrete monoclonal antibody which was determined to be of immunoglobulinclass IgG1 by immunofluorometric assay (IFMA). Hybridoma 9D9F9 wasdeposited with the Leibniz Institute, DSMZ-German Collection ofMicroorganisms and Cell Cultures GmbH, Inhoffenstraße 7B, 38124Braunschweig, Germany, Mar. 23, 1995, and given accession No. ACC 2210.

While specific embodiments have been described above regarding thedisclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the disclosure. Changes andmodifications can be made following ordinary skill in the art withoutdeparting from the disclosure in its broader aspects as defined in thefollowing claims.

1-49. (canceled)
 50. A method to treat cancer in a subject in need ofinhibition of lymphangiogenesis, comprising: a. administering to thesubject, who had been determined to have a dysregulated lymphaticsystem, a therapeutically effective amount of a first monoclonalantibody that targets PD-1 or PD-L1 to antagonize immune checkpointinhibition, thereby inducing an immune modifying effect in the subject;and b. further administering to the subject a therapeutically effectiveamount of a second monoclonal antibody, before or concurrent with theadministration of the first monoclonal antibody, wherein the secondmonoclonal antibody binds to the extracellular domain of VEGFR-3, andwherein the second monoclonal antibody inhibits the lymphangiogenesis inthe subject.
 51. The method of claim 50, wherein the first monoclonalantibody is a chimeric or humanized antibody.
 52. The method of claim50, wherein the first therapeutic antibody targets PD-1.
 53. The methodof claim 50, wherein the first therapeutic antibody targets PD-L1. 54.The method of claim 52, wherein the first monoclonal antibody chosenfrom pembrolizumab, nivolumab, or a combination thereof.
 55. The methodof claim 50, wherein the second monoclonal antibody is a chimeric orhumanized antibody.
 56. The method of claim 50, wherein the secondmonoclonal antibody is administered before the first therapeuticantibody.
 57. The method of claim 50, wherein the second therapeuticantibody is administered concurrently with the first therapeuticantibody.
 58. The method of claim 50, wherein the dysregulated lymphaticsystem is further characterized by one or more conditions chosen fromabnormal lymphatic development, lymphatic proliferation,lymphangiogenesis, impaired lymphatic vessel function, dysregulatedlymphatic vessel function, augmented tumor cell lymphatic infiltration,lymphangitic carcinomatosis, abnormal functioning or homeostaticregulation, lymphatic remodeling, physical pressure upon lymphatics,altered tumoral lymphatic development, altered tumorallymphangiogenesis, and output blockage of lymphatic structures inlymphatic organs.
 59. The method of claim 50, wherein the cancer ischosen form lung cancer, breast cancer, a cancer of the gastrointestinaltract, a cancer of unknown origin, head and neck cancer, bladder cancer,prostate cancer, skin cancer, kidney cancer, a primary brain tumor,ocular tumor, sarcoma, a cancer of primary soft tissue, mesenchymalcancer, bone cancer, a tumor of the lymphatic system, and leukemia. 60.The method of claim 58, wherein the cancer is lung cancer, and themethod further comprises administering one or more drugs chosen fromafatinib dimaleate, alectinib, bevacizumab, carboplatin, ceritinib,crizotinib, docetaxel, doxorubicin, erlotinib, etoposide, everolimus,gefitinib, gemcitabine, mechlorethamine, methotrexate, necitumumab,nivolumab, osimertinib, paclitaxel, paclitaxel albumin-stabilizednanoparticles, pembrolizumab, pemetrexed, ramucirumab, topotecan,vinorelbine, pharmaceutically acceptable salts thereof, and combinationsthereof.
 61. A method to treat cancer in a subject in need of inhibitionof lymphangiogenesis, comprising: a. selecting the subject with acondition of a dysregulated lymphatic system comprisinglymphangiogenesis; b. administering to the subject a therapeuticallyeffective amount of a first monoclonal antibody that targets PD-1 orPD-L1 to antagonize immune checkpoint inhibition, thereby inducing animmune modifying effect in the subject; and c. further administering tothe subject a therapeutically effective amount of a second monoclonalantibody, before or concurrent with the administration of the firstmonoclonal antibody, wherein the second monoclonal antibody binds to theextracellular domain of VEGFR-3, and wherein the second monoclonalantibody inhibits the lymphangiogenesis in the subject.
 62. The methodof claim 61, wherein the first monoclonal antibody is a chimeric orhumanized antibody.
 63. The method of claim 61, wherein the firsttherapeutic antibody targets PD-1.
 64. The method of claim 61, whereinthe first therapeutic antibody targets PD-L1.
 65. The method of claim63, wherein the first monoclonal antibody chosen from pembrolizumab,nivolumab, or a combination thereof.
 66. The method of claim 61, whereinthe second monoclonal antibody is a chimeric or humanized antibody. 67.The method of claim 61, wherein the second monoclonal antibody isadministered before the first therapeutic antibody.
 68. The method ofclaim 61, wherein the second therapeutic antibody is administeredconcurrently with the first therapeutic antibody.
 69. The method ofclaim 61, wherein the dysregulated lymphatic system is furthercharacterized by one or more conditions chosen from abnormal lymphaticdevelopment, lymphatic proliferation, lymphangiogenesis, impairedlymphatic vessel function, dysregulated lymphatic vessel function,augmented tumor cell lymphatic infiltration, lymphangiticcarcinomatosis, abnormal functioning or homeostatic regulation,lymphatic remodeling, physical pressure upon lymphatics, altered tumorallymphatic development, altered tumoral lymphangiogenesis, and outputblockage of lymphatic structures in lymphatic organs.
 70. The method ofclaim 61, wherein the cancer is chosen form lung cancer, breast cancer,a cancer of the gastrointestinal tract, a cancer of unknown origin, headand neck cancer, bladder cancer, prostate cancer, skin cancer, kidneycancer, a primary brain tumor, ocular tumor, sarcoma, a cancer ofprimary soft tissue, mesenchymal cancer, bone cancer, a tumor of thelymphatic system, and leukemia.
 71. The method of claim 70, wherein thecancer is lung cancer, and the method further comprises administeringone or more drugs chosen from afatinib dimaleate, alectinib,bevacizumab, carboplatin, ceritinib, crizotinib, docetaxel, doxorubicin,erlotinib, etoposide, everolimus, gefitinib, gemcitabine,mechlorethamine, methotrexate, necitumumab, nivolumab, osimertinib,paclitaxel, paclitaxel albumin-stabilized nanoparticles, pembrolizumab,pemetrexed, ramucirumab, topotecan, vinorelbine, pharmaceuticallyacceptable salts thereof, and combinations thereof.